Methods and compositions using stearoyl-CoA desaturase to identify triglyceride reducing therapeutic agents

ABSTRACT

The use of screening assays based on the role of human stearoyl-CoA desaturase-1 (“hSCD1”) in human diseases, disorders or conditions relating to serum levels of triglyceride, VLDL, HDL, LDL, total cholesterol, or production of secretions from mucous membranes, monounsaturated fatty acids, wax esters, and the like, is disclosed. Also disclosed are conventions useful in the prevention and/or treatment of such diseases.

This application is a divisional of U.S. application Ser. No.09/792,468, filed 23 Feb. 2001, now U.S. Pat. No. 6,987,001, whichclaims priority of U.S. Provisional Application 60/184,526, filed 24Feb. 2000, U.S. Provisional Application 60/221,697, filed 31 Jul. 2000,and U.S. Provisional Application 60/255,771, filed 15 Dec. 2000, thedisclosures of all of which are hereby incorporated by reference intheir entirety.

FIELD OF THE INVENTION

The present invention relates generally to the field of stearoyl-CoAdesaturase and its involvement in various human diseases. Stearoyl-CoAdesaturase, and the gene encoding it, are useful for identification anddevelopment of therapeutic agents for the treatment of such diseases.

BACKGROUND OF THE INVENTION

Acyl desaturase enzymes catalyze the formation of double bonds in fattyacids derived from either dietary sources or de novo synthesis in theliver. Mammals synthesize four desaturases of differing chain lengthspecificity that catalyze the addition of double bonds at the Δ9, Δ6, Δ5and Δ4 positions. Stearoyl-CoA desaturases (SCDs) introduce a doublebond in the Δ9-position of saturated fatty acids. The preferredsubstrates are palmitoyl-CoA (16:0) and stearoyl-CoA (18:0), which areconverted to palmitoleoyl-CoA (16:1) and oleoyl-CoA (18:1),respectively. The resulting mono-unsaturated fatty acids are substratesfor incorporation into phospholipids, triglycerides, and cholesterolesters.

A number of mammalian SCD genes have been cloned. For example, two geneshave been cloned from rat (SCD1, SCD2) and four SCD genes have beenisolated from mouse (SCD1, 2, 3, and 4). A single SCD gene, SCD1, hasbeen characterized in humans.

While the basic biochemical role of SCD has been known in rats and micesince the 1970's (Jeffcoat R. and James, A T. 1984. Elsevier Science, 4:85-112; de Antueno, R J. 1993. Lipids 28(4)285-290), it has not, priorto this invention, been directly implicated in human disease processes.Studies in non-human animals have obscured our understanding of the roleof SCD in humans due to the well documented differences in thebiochemical processes in different species. In rodents, for example,lipid and cholesterol metabolism is particularly obscured by the absenceof Cholesterol Ester Transport Protein (CETP) (see Foger, B. et al.1999. J. Biol. Chem. 274(52) 36912).

Further, the existence of multiple SCD genes in mice and rats addsadditional complexity to determining the specific role of each of thesegenes in disease processes. Differences in tissue expression profiles,substrate specificity, gene regulation and enzyme stability may beimportant in elucidating which SCD gene plays the dominant role in eachdisorder. Most previous SCD studies assess SCD gene function bymeasuring mRNA levels or by measuring levels of monounsaturated fattyacids as an indirect measure of SCD enzyme activity. In both these casesthis analysis may be misleading. In the latter method it has beenparticularly misleading and difficult to discern the relativecontribution of SCD1 to the plasma desaturation index (the ratio ofmonounsaturated fatty acids to saturated fatty acids of a specific chainlength) due to the fact that multiple SCD enzymes may contribute to theproduction of monounsaturated fatty acids. Prior to this invention, therelative contributions of the multiple SCD isoforms to the desaturationindex was unknown. In summary, previous studies have not differentiatedwhich SCD isoforms play a major role in the total desaturase activity asmeasured by the desaturation index.

Recent work in in vitro chicken hepatocyte cell culture relates delta-9desaturase activity to impaired triacylglycerol secretion (Legrand, P.and Hermier, D. (1992) Int. J. Obesity 16, 289-294; Legrand, P.,Mallard, J., Bernard-Griffiths, M. A., Douaire, M., and Lemarchal, P.Comp. Biochem. Physiol. 87B, 789-792; Legrand, P., Catheline, D.,Fichot, M.-C., Lemarchal, P. (1997) J. Nutr. 127, 249-256). This workdid not distinguish between isoforms of delta-9 desaturase that mayexist in the chicken, once again failing to directly implicate aspecific SCD enzyme to account for a particular biological effect, inthis case, impaired triglyceride secretion.

Nor does this in vitro work correlate well to humans because substantialdifferences exist between chicken and human lipoprotein metabolism invivo. Such differences include the presence, in chicken, of entirelydifferent lipoproteins, such as vitellogenin, and distinct processessuch as the massive induction of hepatic triglyceride synthesis duringovulation. Other differences such as the type of lipoproteins used forcholesterol transport and the process of secretion of dietarytriglyceride in chylomicrons are well documented. These majordifferences between avians and mammals mean that extrapolation from theavians to mammals in the area of triglyceride metabolism must beconsidered provisional pending confirmation in humans.

Two other areas of background art form an important basis to the instantinvention. Firstly, this invention relates to cholesterol and lipidmetabolism, which in humans has been intensely studied. Sincecholesterol is highly apolar, it is transported through the bloodstreamin the form of lipoproteins consisting essentially of a core of apolarmolecules such as cholesterol ester and triglyceride surrounded by anenvelope of amphipathic lipids, primarily phospholipids. In humans,approximately 66% of cholesterol is transported on low densitylipoprotein (LDL) particles, about 20% on high density lipoprotein (HDL)particles, and the remainder on very low density lipoprotein (VLDL)particles. An excellent reference to the basic biochemistry ofcholesterol metabolism in humans and other organisms is found at Biologyof Cholesterol. Ed. Yeagle, P. CRC Press, Boca Raton, Fla., 1988.

Secondly, this invention takes advantage of new findings from the Asebiamouse (Gates, et al. (1965) Science. 148:1471-3). This mouse is anaturally occurring genetic variant mouse that has a well known defectin sebaceous glands, resulting in hair loss and scaly skin. The Asebiamouse has recently been reported to have a deletion in SCD1 resulting inthe formation of an early termination site in exon 3 of the SCD1 gene.Animals homozygous for this mutation, or a distinct deletion allelewhich encompasses exons 1-4, do not express detectable amounts of thewild-type SCD1 mRNA transcript (November 1999. Nature Genetics. 23:268et seq.; and PCT patent publication WO 00/09754)]. Since the full extentof this naturally occurring deletion is unknown, it is also unknown ifother genes neighboring SCD1, or elsewhere in the genome, could also beinvolved in the Asebia phenotype. In order to specifically study theactivity of SCD1 in these disease processes, a specific SCD1 knockoutmouse is required. The prior work on this variant has focused on therole of this mutation in skin disorders and not on triglyceride or VLDLmetabolism.

It is an object of the instant invention to identify diseases anddisorders that are linked specifically to SCD1 biological activity inhumans, and in a preferred embodiment, diseases and disorders oftriglyceride metabolism. It is a further object to develop screeningassays to identify and develop drugs to treat those diseases, disordersand related conditions. Further, it is an object of this invention toprovide compositions for use in treating these disease, disorders andrelated conditions.

BRIEF SUMMARY OF THE INVENTION

This invention discloses, for the first time, the role of humanstearoyl-CoA desaturase-1 (“hSCD1”) in a wide range of human diseasesand disorders. In particular, SCD1 biological activity in humans isdirectly related to serum levels of triglycerides and VLDL. In addition,SCD1 biological activity also affects serum levels of HDL, LDL, and/ortotal cholesterol, reverse cholesterol transport, and the production ofsecretions from mucous membranes, monounsaturated fatty acids, waxesters, and/or the like.

It is an object of the present invention to provide a process orscreening assay for identifying, from a library of test compounds, atherapeutic agent which modulates the biological activity of said humanstearoyl-CoA desaturase (hSCD1) and is useful in treating a humandisorder or condition relating to serum levels of triglyceride or VLDL.Preferably, the screening assay identifies inhibitors of hSCD1 whichlower serum triglyceride levels and provide an importantcardioprotective benefit for humans.

It is also an object of the present invention to provide a process orscreening assay for identifying, from a library of test compounds, atherapeutic agent which modulates the biological activity of said humanstearoyl-CoA desaturase (hSCD1) and is useful in treating a humandisorder or condition relating to serum levels of HDL, LDL, and/or totalcholesterol, reverse cholesterol transport or the production ofsecretions from mucous membranes, monounsaturated fatty acids, waxesters, and/or the like

In one aspect, the present invention relates to vectors comprising humanstearoyl-CoA desaturase (hSCD1) genes and promoter sequences and torecombinant eukaryotic cells, and cell lines, preferably mammaliancells, and most preferably human cells, and cell lines, transfected soas to comprise such vectors and/or said polynucleotides and wherein saidcells express hSCD1. Disclosed herein is the full length promotersequence for hSCD1, SEQ ID. No. 1.

It is also an object of the present invention to provide agents capableof modulating the activity and/or expression of human stearoyl-CoAdesaturase 1 (hSCD1) as disclosed herein, especially where saidmodulating ability was first determined using an assay comprising hSCD1biological activity or a gene encoding hSCD1. Pharmaceuticalcompositions comprising such agents are specifically contemplated.

It is a still further object of the present invention to provide agentswherein said agent is useful in treating, preventing and/or diagnosing adisease or condition relating to hSCD1 biological activity.

It is a yet further object of the present invention to provide a processfor preventing or treating a disease or condition in a patient afflictedtherewith comprising administering to said patient a therapeutically orprophylactically effective amount of a composition as disclosed herein.

In a pharmacogenomic application of this invention, an assay is providedfor identifying cSNPs (coding region single nucleotide polymorphisms) inhSCD1 of an individual which are associated with human disease processesor response to medication.

In other aspects, the present invention also provides a process fordiagnosing a disease or condition in a patient, commonly a human being,suspected of being afflicted therewith, or at risk of becoming afflictedtherewith, comprising obtaining a tissue sample from said patient anddetermining the level of activity of hSCD1 in the cells of said tissuesample and comparing said activity to that of an equal amount of thecorresponding tissue from a patient not suspected of being afflictedwith, or at risk of becoming afflicted with, said disease or condition.

In other aspects, the present invention also provides a process fordiagnosing a disease or condition in a patient, commonly a human being,suspected of being afflicted therewith, or at risk of becoming afflictedtherewith, comprising obtaining a tissue sample from said patient andidentifying mutations in the hSCD1 gene in the cells of said tissuesample and comparing said gene to that of a corresponding tissue from apatient not suspected of being afflicted with, or at risk of becomingafflicted with, said disease or condition.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Generation of SCD1 null mice (A) Targeting strategy for SCD1. Apartial map of the genomic locus surrounding the Scd1 locus is shown.Homologous recombination resulted in the replacement of exons 1-6 by neo7 gene. Gene-targeting events were verified by Southern blot analysisusing EcoRI and probe A or B or by PCR analysis. (B) PCR analysisdemonstrating SCD−/− mice. In breeding heterozygotes, wild-type,heterozygotes and homozygotes were born in Mendelian fashion(+/+:+/−:−/−=21:43:20 x²=0.395). (C) Northern blot analysis. 20 μg oftotal RNA was isolated from the liver and subjected to Northern blotanalyses. Blots were probed with a mouse SCD1 and 2 cDNA fragments. (D)Immunoblot analysis of liver showed the absence of immunoreactive SCD inSCD1−/− mice, whereas SCD1 protein was detected in liver tissue fromboth wild-type and heterozygote mice in a manner dependent on genedosage. (E) Liver SCD activity was abolished in SCD−/− mice. Asmentioned above, heterozygotes present an intermediate phenotype whencompared to wild-type and null littermates. Enzyme activity isrepresented as nanomoles of substrate desaturated per milligram ofprotein per minute. Data are denoted as the mean ±SD (n=3).

FIG. 2. Plasma lipoprotein profiles in SCD1 Knock-out and Asebia MaleMice. The top two panels depict the triglyceride content of thelipoprotein fractions, the bottom two panels depict the cholesterolcontent of the lipoprotein fractions.

FIG. 3. VLDL-triglyceride levels in Asebia (SCD1−/−) and SCD1+/− mice.Plasma lipoproteins were separated by fast performance liquidchromatography and the distribution of triglycerides among lipoproteinsin the various density fractions of the mice (n=3) were measured. SCD−/−(open circles), SCD1+/− (filled circles). The lipoprotein peaks forVLDL, LDL and HDL are indicated.

FIG. 4. Ratio of monounsaturated to saturated fatty acid in mouse plasma(the desaturation index) decreases in a manner directly proportional tothe level of SCD activity I. Comparison of SCD1 knock-out and asebiamice to their respective controls.

FIG. 5 shows a linear regression analysis using a human data set. Theratio of 18:1/18:0 showed a significant relationship to TG levels(r₂=0.39, p<0.0001) (Panel A), as well as a significant correlations toHDL levels (r²=0.12, p=0.0006) (Panel B). The experimental details arefurther described in Example 2.

FIG. 6 shows a linear regression analysis indicating a weak relationshipbetween the relative level of 16:1/16:0 to plasma TG levels was observed(r²=0.05, p=0.03). Experimental details are in Example 2.

FIG. 7 shows a linear regression analysis of those individuals with ahigh HDL phenotype (>90^(th) percentile). These individuals demonstrateda significant relationship between the 18:1/18:0 ratio and TG levels(r²=0.40, p<0.005).

FIG. 8 shows an observed relationship between 18:1/18:0 and TG levelswas observed in a family (HA-1) that segregates a high HDL phenotype.Using linear regression analysis, a significant relationship between18:1/18:0 and TG was observed (r²=0.36, p=0.005 (Panel A)). Panel Bshows a significant relationship between 18:1/18:0 ratio and HDL levelsin this family (r²=0.32, p=0.009).

FIG. 9 shows the relationship observed between the 18:1/18:0 ratio andTG levels (r²=0.49, p=0.0009) when only persons with low HDL (<5^(th)percentile) are considered.

FIG. 10 shows an analysis of a family (NL-001), which segregated a lowHDL phenotype of unknown genetic etiology and tended towards therelationships observed in FIGS. 5-9.

FIG. 11 shows an analysis of family NL-0020 which segregated an ABCA1mutation and tended towards the relationships noted in FIGS. 5-9.

FIG. 12 is a plasma fatty acid analysis showing the relationship betweenthe 18:1/18:0 ratio and TG levels (r²=0.56, p=0.02) (Panel A), HDLlevels (r2=0.64, p=0.0095) (Panel B) and total cholesterol levels(r2=0.50, p=0.03) in nine persons with Familial Combined Hyperlipidemia(FCHL) (Panel C).

FIG. 13 is a plasma fatty acid analysis showing a significantly elevated18:1/18:0 ratio in hyperlipidemic mice (HcB-19) when compared tounaffected controls of the parental strain (C3H).

FIG. 14. Location of regulatory sequences and binding sites inhomologous region of the mouse SCD1 and human SCD1 promoter and5′-flanking regions. The top scale denotes the position relative to thetranscriptional start site. Important promoter sequence elements areindicated.

FIG. 15. Human HepG2 cells cultured and treated with a range of doses ofarachidonic acid, DHA or 10 μg/ml cholesterol or EPA as indicated. TotalmRNA was isolated and quantified.

FIG. 16. TLC of lipid extracts from skin (A and B) and eyelids (C and D)of wild-type, heterozygotes and SCD−/− mice. Total lipids were extractedfrom eyelids of wild-type, heterozygotes and SCD−/− mice. Lipid extractswere pooled and analyzed by high performance TLC (HPTLC, A and C; hexaneether/ether/acetic acid=90:25:1, B and D; Benzene:hexane; 65:35). Sameamounts of lipid extract (from 0.5 mg of eyelid) were subjected in eachlane. Each lane represents lipids from eyelids of two mice.

FIG. 17 shows an assay for SCD1 desaturase activity by quantifyingtransfer of 3H from stearate to water. The figure shows a time course of3H-water production at room temperature. Microsomes from wild typelivers were used for this experiment. A turnover number for SCD1activity under these conditions was estimated at 2 nmol/min/mg protein,which is about half that observed at 37° C.

FIG. 18 shows validation that the assay monitors specificallySCD1-dependent desaturation of stearoyl-CoA. Livers were collected fromwild type and SCD1 knockout mice following 3 days on a high carbohydratefat-free diet (this induces SCD1 expression in liver by about 50-fold),homogenized and a portion used for microsome purification. 3H-waterproduction was determined for 15 minutes at RT (room temperature) inboth homogenate and microsome preparations at equivalent proteinconcentration, followed by quenching the reaction with acid and usingcharcoal to separate substrate from product. Quenching the sample withacid prior to the addition of substrate offers a blank, or controlcontaining background radioactivity without any desaturation reaction.The figure shows that desaturation activity in microsomes is greatlyenriched compared to the homogenate for wild type livers, while themicrosomes from the −/− SCD1 knockout animal has very little activity.The “window”, or SCD1-dependent desaturation in this assay, is a highlyvisible and significant 160-fold difference between wild type and SCD1knockout microsomes.

FIG. 19 shows inhibition of SCD1 with 3 known fatty acid inhibitors.Microsomes from wild type mice were used to test the effectiveness ofthree known inhibitors of SCD1: conjugated linoleic acid (CLA), 9-thiastearic acid (9-thia) and sterculic acid (SA). Panel A shows that whenadded as the free fatty acid none were effective to suppressSCD1activity. However, panel B shows that if pre-conjugated to CoA (doneby incubating the microsomes with CoA and ATP prior to the addition of³H-stearoyl CoA) the three inhibitors show graded inhibition of SCD1with sterculic acid suppressing nearly 100% of the activity for thepreincubation condition. This experiment establishes that SCD1 activitycan be inhibited with known inhibitors but they appear to requireconjugation with CoA. An important use of this screening assay is tofind small molecules that are potent inhibitors of SCD1 biologicalactivity without conjugation to CoA.

FIG. 20 (A) Demonstration that stearoyl-CoA mass limits the kinetics andmagnitude of the ³H-production signal we are taking as a measure ofSCD1-dependent desaturation. This experiment is essentially a repeat ofthat shown in FIG. 17 with the exception that at 30 mins an additionalaliquot of stearoyl-CoA mass/radioactivity was added, resulting in thesecond exponential production of ³H signal. This shows that the amountof stearyol-CoA limits the reaction as expected for SCD1-catalyzeddesaturation. (B) Demonstration that the experiment is adaptable to highthroughput. All previous experiments were done where the total reactionvolume was 1.1 ml (0.2 ml reaction buffer containing microsomes, 0.2 ml6% PCA to quench the reaction and 0.7 ml 10% charcoal solution tosediment the unreacted substrate). The experiment illustrated in B wasdone with a total reaction volume of 0.31 ml (0.1 ml reaction bufferwith microsomes, 0.01 ml 60% PCA to quench and 0.2 ml 10% charcoal tosediment).

DEFINITIONS

“Isolated” in the context of the present invention with respect topolypeptides or polynucleotides means that the material is removed fromits original environment (e.g., the natural environment if it isnaturally occurring). For example, a naturally-occurring polynucleotideor polypeptide present in a living organism is not isolated, but thesame polynucleotide or polypeptide, separated from some or all of theco-existing materials in the natural system, is isolated. Suchpolynucleotides could be part of a vector and/or such polynucleotides orpolypeptides could be part of a composition, and still be isolated inthat such vector or composition is not part of its natural environment.The polypeptides and polynucleotides of the present invention arepreferably provided in an isolated form, and preferably are purified tohomogeneity.

The nucleic acids and polypeptide expression products disclosedaccording to the present invention, as well as expression vectorscontaining such nucleic acids and/or such polypeptides, may be in“enriched form.” As used herein, the term “enriched” means that theconcentration of the material is at least about 2, 5, 10, 100, or 1000times its natural concentration (for example), advantageously 0.01%, byweight, preferably at least about 0.1% by weight. Enriched preparationsof about 0.5%, 1%, 5%, 10%, and 20% by weight are also contemplated. Thesequences, constructs, vectors, clones, and other materials comprisingthe present invention can advantageously be in enriched or isolatedform.

The polynucleotides, and recombinant or immunogenic polypeptides,disclosed in accordance with the present invention may also be in“purified” form. The term “purified” does not require absolute purity;rather, it is intended as a relative definition, and can includepreparations that are highly purified or preparations that are onlypartially purified, as those terms are understood by those of skill inthe relevant art. For example, individual clones isolated from a cDNAlibrary have been conventionally purified to electrophoretichomogeneity. Purification of starting material or natural material to atleast one order of magnitude, preferably two or three orders, and morepreferably four or five orders of magnitude is expressly contemplated.Furthermore, claimed polypeptide which has a purity of preferably0.001%, or at least 0.01% or 0.1%; and even desirably 1% by weight orgreater is expressly contemplated.

The term “coding region” refers to that portion of a gene which eithernaturally or normally codes for the expression product of that gene inits natural genomic environment, i.e., the region coding in vivo for thenative expression product of the gene. The coding region can be from anormal, mutated or altered gene, or can even be from a DNA sequence, orgene, wholly synthesized in the laboratory using methods well known tothose of skill in the art of DNA synthesis.

In accordance with the present invention, the term “nucleotide sequence”refers to a heteropolymer of deoxyribonucleotides (for DNA) orribonucleotides (for RNA). Generally, DNA segments encoding the proteinsprovided by this invention are assembled from cDNA fragments and shortoligonucleotide linkers, or from a series of oligonucleotides, toprovide a synthetic gene which is capable of being expressed in arecombinant transcriptional unit comprising regulatory elements derivedfrom a microbial or viral operon.

The term “expression product” means that polypeptide or protein that isthe natural translation product of the gene and any nucleic acidsequence coding equivalents resulting from genetic code degeneracy andthus coding for the same amino acid(s).

The term “fragment,” when referring to a coding sequence, means aportion of DNA comprising less than the complete coding region whoseexpression product retains essentially the same biological function oractivity as the expression product of the complete coding region.

The term “primer” means a short nucleic acid sequence that is pairedwith one strand of DNA and provides a free 3′OH end at which a DNApolymerase starts synthesis of a deoxyribonucleotide chain.

The term “promoter” means a region of DNA involved in binding of RNApolymerase to initiate transcription, and includes all nucleotidesupstream (5′) of the transcription start site.

As used herein, reference to a DNA sequence includes both singlestranded and double stranded DNA. Thus, the specific sequence, unlessthe context indicates otherwise, refers to the single strand DNA of suchsequence, the duplex of such sequence with its complement (doublestranded DNA) and the complement of such sequence.

The present invention further relates to a polypeptide which has thededuced amino acid sequence, as well as fragments, analogs andderivatives of such polypeptide.

The terms “fragment,” “derivative” and “analog” when referring to thepolypeptide, means a polypeptide which retains essentially the samebiological function or activity as such polypeptide. Thus, an analogincludes a proprotein which can be activated by cleavage of theproprotein portion to produce an active mature polypeptide. Suchfragments, derivatives and analogs must have sufficient similarity tothe SCD1 polypeptide so that activity of the native polypeptide isretained.

The polypeptide of the present invention may be a recombinantpolypeptide, a natural polypeptide or a synthetic polypeptide, and ispreferably a recombinant polypeptide.

The fragment, derivative or analog of the SCD1 polypeptide may be (i)one in which one or more of the amino acid residues are substituted witha conserved or non-conserved amino acid residue (preferably a conservedamino acid residue) and such substituted amino acid residue may or maynot be one encoded by the genetic code, or (ii) one in which one or moreof the amino acid residues includes a substituent group, or (iii) one inwhich the mature polypeptide is fused with another compound, such as acompound to increase the half-life of the polypeptide (for example,polyethylene glycol), or (iv) one in which the additional amino acidsare fused to the mature polypeptide, such as a leader or secretorysequence or a sequence which is employed for purification of the maturepolypeptide or a proprotein sequence. Such fragments, derivatives andanalogs are deemed to be within the scope of those skilled in the artfrom the teachings herein.

As known in the art “similarity” between two polypeptides is determinedby comparing the amino acid sequence and its conserved amino acidsubstitutes of one polypeptide to the sequence of a second polypeptide.

In accordance with the foregoing, the present invention also relates toan isolated stearoyl-CoA desaturase encoded by the isolatedpolynucleotide of the invention.

Fragments or portions of the polypeptides of the present invention maybe employed for producing the corresponding full-length polypeptide bypeptide synthesis; therefore, the fragments may be employed asintermediates for producing the full-length polypeptides. Fragments orportions of the polynucleotides of the present invention may be used tosynthesize full-length polynucleotides of the present invention.

As used herein, the terms “portion,” “segment,” and “fragment,” whenused in relation to polypeptides, refer to a continuous sequence ofresidues, such as amino acid residues, which sequence forms a subset ofa larger sequence. For example, if a polypeptide were subjected totreatment with any of the common endopeptidases, such as trypsin orchymotrypsin, the oligopeptides resulting from such treatment wouldrepresent portions, segments or fragments of the starting polypeptide.When used in relation to polynucleotides, such terms refer to theproducts produced by treatment of said polynucleotides with any of thecommon endonucleases.

DETAILED SUMMARY OF THE INVENTION

The present invention relates to the activity of human stearoyl-CoAdesaturase-1 in human disease processes. In accordance therewith,compounds that specifically modulate human stearoyl-CoA desaturase-1activity or expression level are useful in the treatment of a humandisorder or condition relating to serum levels of triglyceride or VLDL,and provide an important cardioprotective benefit when administered tohumans. Compounds that modulate hSCD1 activity or expression are alsouseful for modulating serum levels of HDL, LDL, and/or totalcholesterol, and/or reverse cholesterol transport. Finally, compoundsthat modulate hSCD1 activity or expression are also useful formodulating the production of secretions from mucous membranes,monounsaturated fatty acids, wax esters, and the like.

The SCD1 Gene and Protein

Human Stearoyl-CoA Desaturase-1 (also called SCD1, hSCD and hSCD1) hasbeen identified with the full cDNA sequence first released to GenBank asGenBank Accession Y13647 (also NM005063) dated Jun. 6, 1997. Furtherdescriptions of SCD1, including partial promoter sequences can be foundon GenBank under the following accession numbers:gb|AF097514.1|AF097514; dbj|AB032261.1|AB032261; gb|AF116616.1|AF116616;ref|XM_(—)005719.1|; gb|AF113690.1 |AF113690; and gb|S70284.1|S70284

In one aspect the present invention relates to uses of an isolatedpolynucleotide comprising a non-genomic polynucleotide having at least90% identity, preferably 95% identity, most preferably at least a 98%identity to the sequence of human stearoyl-CoA reductase-1, especiallywhere said sequences are the same and including any of the complementsof any of the foregoing.

The full promoter sequence of hSCD1 is SEQ ID. No. 1 and FIG. 14illustrates the functional elements conserved between the mouse andhuman SCD1 promoter regions.

In one aspect the present invention relates to uses of an isolatedpolypeptide having at least 90% identity, preferably 95% identity, mostpreferably at least a 98% identity to human stearoyl-CoA reductase-1,especially where said sequences are the same. The polypeptide sequencehas been previously disclosed and can be found at the followingSwissProtein database accession series: ACCESSION No. O00767; PIDg3023241; VERSION O00767 GI:3023241; DBSOURCE: swissprot: locusACOD_HUMAN, accession O00767. Alternatively, the polypeptide sequencecan be determined from the cDNA sequence references provided above.

SCD1 in Human Disease Processes

As disclosed herein, a number of human diseases and disorders are theresult of aberrant SCD1 biological activity and may be ameliorated bymodulation of SCD1 biological activity using therapeutic agents.

The most significant teaching of the present disclosure relates to therole of SCD1 in modulating serum triglyceride and VLDL levels in humans.Two major findings are established herein. Firstly, Example 1 belowshows that the lipoprotein profiles of SCD1 knock-out mice demonstrate a65% reduction in serum triglyceride and VLDL levels. These correspondwith the lipoprotein profiles of Asebia mice which are also includedherein. The lipoprotein profiles of both the Asebia and the SCD1knock-out mouse were not previously known but due to the targeted andspecific nature of the engineered mutation, the correlation of SCD1activity and serum triglyceride levels can be drawn with certainty.While other SCD isoforms may play a role in triglyceride levels, thisdata indicates that SCD1, specifically, plays the major role in thisprocess.

There are significant differences in lipoprotein metabolism betweenmouse and humans, and while the foregoing data are convincing in themouse for a major and specific role for SCD1 in modulation oftrigylceride levels, this still needed confirmatory experiments inhumans. The second major finding, therefore, presented in Example 2,below, demonstrates a significant correlation between SCD activity inhumans and levels of serum triglycerides. It has thus been discoveredthat SCD1 biological activity in humans is directly related to levels ofserum triglycerides.

In accordance with the present invention, the Asebia mouse phenotype(first described by Gates, et al. (1965) Science. 148:1471-3) shows amajor and significant alteration in serum lipoprotein profile includinga large reduction in triglyceride and VLDL levels. In addition, theseanimals have a large decrease in liver content of cholesterol esters. Inaccordance therewith, effective inhibition of SCD1 activity would leadto a reduction in triglyceride levels, due to decreased availability ofmonounsaturated fatty acids. Monounsaturated fatty acids are thepreferred substrate for the enzyme responsible for triglyceride (TG)synthesis from fatty acids and glycerol phosphate (viz., glycerolphosphate acyl transferase (GPAT)).

Also in accordance with the disclosure herein, increased esterificationof cholesterol prevents the toxic accumulation of free cholesterol inliver, and the increase in the availability of cholesterol esters andtriglycerides also facilitates their secretion in the form of VLDL.Increased cholesterol esterification in macrophages may also enhance theformation of foam cells and thereby contribute to atherosclerotic lesiondevelopment. Thus, the inhibition of SCD activity may have the addedeffect of reducing the level of VLDL particles in the bloodstream andinhibiting atherosclerosis.

Further in accordance with the present invention, inhibition of SCD1 isalso advantageous in increasing the formation of HDL at peripheraltissues. In a healthy individual, cellular cholesterol is predominantlyin the esterified form, with low levels of free cholesterol.Acyl-CoA:cholesterol acyltransferase (ACAT) is the enzyme responsiblefor esterifying cholesterol using monounsaturated fatty acyl-CoA's as apreferred substrate. SCD generates the monounsaturated products, whichare then available for cholesterol esterification by ACAT. The increasedflux of free cholesterol out of cells and through HDL is thought to betherapeutically beneficial because it would signify enhanced “reversecholesterol transport” (RCT).

Inhibition of SCD1 is also useful in increasing reverse cholesteroltransport (RCT) without necessarily raising the serum HDL level. SerumHDL level is a surrogate marker for the process of RCT, which in factpreferably is measured by the overall flux of cholesterol fromperipheral tissues to the liver. The invention identifies modulators ofSCD1 biological activity as effective therapeutic agents for increasingRCT. RCT can be directly measured, for example, by injectingradiolabelled cholesterol ether incorporated into HDL particles directlyinto the blood, and measuring the clearance rate (the rate at which itis taken-up into the liver of an organism).

In accordance with the present invention, it has been found thatmodulation of SCD1 activity in the liver and other tissues results in anincrease in SR-B1, a liver receptor which removes HDL from thecirculation, thus increasing RCT with less obvious effects on HDL levelsin the blood. The linkage between SCD1 biological activity and SR-B1mRNA expression has not previously been identified. Previous work hasestablished that SR-B1-overexpressing mice are cardioprotected,demonstrating reduced atherogenesis and reduced cardiovascular disease.This understanding also suggests for the first time that certaintherapeutic agents, such as inhibitors of SCD1 biological activity, mayincrease RCT without any obvious changes on HDL levels. This is achievedby obtaining a balanced increase in both HDL formation in peripheraltissues and HDL removal by the liver.

The experiment compared SR-B1 mRNA expression in the liver of +/+ versus−/− SCD1mice (strains as described in the Examples below). Whenexpressed relative to the +/+ animal on chow diet the results show thefollowing for changes in ABCA1 and SR-B1 mRNA levels:

genotype diet ABC1 SR-B1 +/+ Chow 1 1 −/− Chow 0.7 11 +/+ Hi Cholesterol1.1 27 −/− Hi Cholesterol 0.4 27

The changes in ABC1 are not significant while those shown for SR-B1 on achow diet are. An increase in SR-B1 expression indicates increased flux,or RCT, of cholesterol to the liver and may explain why there is noobservation of elevated HDL-C in the plasma of the −/− SCD1 mouse.Increased RCT is further confirmed by the finding that −/− animals onhigh cholesterol diet have a gall bladder roughly 10-times the size ofthe +/+ animals and which are engorged with bile. These observations areconsistent with increased removal of cholesterol by the liver, henceincreased RCT. Further, the apparently identical increase in SR-B1 in+/+ and −/− mice may not reflect an identical phenotype or biologicalprocess in these animals.

Inhibition of SCD expression may also affect the fatty acid compositionof membrane phospholipids, as well as triglycerides and cholesterolesters. The fatty acid composition of phospholipids ultimatelydetermines membrane fluidity, while the effects on the composition oftriglycerides and cholesterol esters can affect lipoprotein metabolismand adiposity.

The present invention also relates to the involvement of SCD1 in otherhuman disorders or conditions relating to serum levels of HDL, LDL, andtotal cholesterol as well as the role of SCD1 in other human disordersor conditions relating to the production of secretions from mucousmembranes, monounsaturated fatty acids, wax esters, and the like. Theinvention encompasses modulators of SCD1 that are useful for treatingthese disorders.

Previous work not using human subjects has shown that aberrant SCDbiological activity in those organisms (but not specifying which isoformof SCD was responsible) may be implicated in various skin diseases, aswell as such diverse maladies as cancer and multiple sclerosis,non-insulin-dependent diabetes mellitus, hypertension, neurologicaldiseases, skin diseases, eye diseases, immune disorders, and cancer.Modulators discovered using the processes of the present invention wouldthereby also find use in treating those diseases and disorders in humansubjects.

In Example 4, transcription regulating proteins for SCD1 are identified.These proteins are targets for compounds that increase or decrease SCD1expression in cells, thereby influencing, either positively ornegatively, SCD1 biological activity of cells. PPAR-gamma and SREBP areexamples. Compounds which are known to act through such transcriptionregulators may now be identified as relevant for treating the SCD1related diseases and disorders now identified in humans.

Screening Assays

The present invention provides screening assays employing the hSCD1 geneand/or protein for use in identifying therapeutic agents for use intreating a disorder or condition relating to serum levels oftriglyceride, VLDL, HDL, LDL, total cholesterol, reverse cholesteroltransport, the production of secretions from mucous membranes,monounsaturated fatty acids, wax esters, and the like.

“SCD1 Biological Activity”

“SCD1 biological activity” as used herein, especially relating toscreening assays, is interpreted broadly and contemplates all directlyor indirectly measurable and identifiable biological activities of theSCD1 gene and protein. Relating to the purified SCD1 protein, SCD1biological activity includes, but is not limited to, all thosebiological processes, interactions, binding behavior, binding-activityrelationships, pKa, pD, enzyme kinetics, stability, and functionalassessments of the protein. Relating to SCD1 biological activity in cellfractions, reconstituted cell fractions or whole cells, these activitiesinclude, but are not limited the rate at which the SCD introduces acis-double bond in its substrates palmitoyl-CoA (16:0) and stearoyl-CoA(18:0), which are converted to palmitoleoyl-CoA (16:1) and oleoyl-CoA(18:1), respectively, and all measurable consequences of this effect,such as triglyceride, cholesterol, or other lipid synthesis, membranecomposition and behavior, cell growth, development or behavior and otherdirect or indirect effects of SCD1 activity. Relating to SCD1 genes andtranscription, SCD1 biological activity includes the rate, scale orscope of transcription of genomic DNA to generate RNA; the effect ofregulatory proteins on such transcription, the effect of modulators ofsuch regulatory proteins on such transcription; plus the stability andbehavior of mRNA transcripts, post-transcription processing, mRNAamounts and turnover, and all measurements of translation of the mRNAinto polypeptide sequences. Relating to SCD1 biological activity inorganisms, this includes but is not limited biological activities whichare identified by their absence or deficiency in disease processes ordisorders caused by aberrant SCD1 biological activity in thoseorganisms. Broadly speaking, SCD1 biological activity can be determinedby all these and other means for analyzing biological properties ofproteins and genes that are known in the art.

The screening assays contemplated by the present invention may alsoemploy isoforms of SCD from humans or other organisms that demonstratesimilar biological activity as hSCD1 so long as they succeed inidentifying therapeutic agents for human diseases. The functionalequivalency of delta-9 desaturases from vertebrates has been recognizedby those in the art. Consequently, specific embodiments of the presentinvention may employ one or more functionally equivalent delta-9desaturase enzymes from another vertebrate species to identifytherapeutic agents useful for humans. Functionally equivalentdesaturases include all of the mouse, rat, cow, pig or chicken SCDsidentified above, in addition to the genes identified at the UniGeneCluster Hs. 119597 SCD for Stearoyl-CoA desaturase (delta-9-desaturase).See also LocusLink: 6319; OMIM: 604031 or HomoloGene: Hs. 119597. Otherknown delta-9 desaturases include pig: 002858 (swiss-prot); and cow:AF188710 (NCBI, [6651449, Genbank])

Selected model Organism Protein Similarities

(organism, protein reference and percent identity and length of alignedamino acid (aa) region)

H. sapiens: SP: O00767- 100%/358 aa  M. musculus: PIR: A32115- 83%/357aa R. norvegicus: SP: P07308- 84%/357 aa D. melanogaster: PID: g1621653-57%/301 aa C. elegans: PID: g3881877- 52%/284 aa S. cerevisiae: PID:e243949- 36%/291 aa B. Taurus O02858 85%/359 aa S. Scrofa 665144986%/334 aaDesign and Development of SCD Screening Assays

The present disclosure facilitates the development of screening assaysthat may be cell based, cell extract (i.e. microsomal assays), cell free(i.e. transcriptional) assays, and assays of substantially purifiedprotein activity. Such assays are typically radioactivity orfluorescence based (i.e. fluorescence polarization or fluorescenceresonance energy transfer or FRET), or they may measure cell behavior(viability, growth, activity, shape, membrane fluidity, temperaturesensitivity etc). Alternatively, screening may employ multicellularorganisms, including genetically modified organisms such as knock-out orknock-in mice, or naturally occurring genetic variants. Screening assaysmay be manual or low throughput assays, or they may be high throughputscreens which are mechanically/robotically enhanced.

The aforementioned processes afford the basis for screening processes,including high throughput screening processes, for determining theefficacy of potential therapeutic and diagnostic drugs for treating thediseases described herein, preferably diseases in which increased ordecreased activity or expression of stearoyl-CoA desaturase (hSCD1 ofthe invention) plays a key role in mediating such disease.

As such this invention relates to a method for identifying, such as froma library of test compounds, a therapeutic agent which is useful inhumans for the treatment of a disorder or condition relating to serumlevels of triglyceride, VLDL, HDL, LDL, total cholesterol or productionof secretions from mucous membranes, monounsaturated fatty acids, waxesters, and the like, comprising

-   -   a) providing a screening assay having SCD1 biological activity;    -   b) contacting said screening assay with a test compound; and    -   c) subsequently measuring said biological activity;    -   wherein a test compound which modulates said biological activity        is said therapeutic agent, or an analog thereof.

In one aspect, the present invention relates to a process foridentifying, from a library of test compounds, a therapeutic agent whichis useful in humans for the treatment of a disorder or conditionrelating to serum levels of triglyceride or very low density lipoprotein(VLDL) comprising

-   -   a) providing a screening assay having stearoyl-Coenzyme A        desaturase type 1 (SCD1) biological activity as a component        thereof;    -   b) contacting said SCD1 activity with a test compound;    -   c) administering to a human a compound found to modulate said        activity in (b); and    -   (d) detecting a change in serum level of triglyceride or VLDL in        said human following said administering;    -   thereby identifying an agent useful in the treatment of a        disorder or condition relating to serum levels of triglyceride        or very low density lipoprotein (VLDL).

In one embodiment, said agent is an antagonist or inhibitor of SCD1biological activity. In another specific embodiment thereof, said agentis an agonist of SCD1 biological activity.

In another embodiment, where said modulator is an inhibitor, saidinhibitor does not substantially inhibit the biological activity in ahuman of a delta-5 desaturase, delta-6 desaturase or fatty acidsynthetase

In one embodiment of the present invention, the assay process furthercomprises the step of assaying said therapeutic agent to further selectcompounds which do not substantially inhibit in a human the activity ofdelta-5 desaturase, delta-6 desaturase or fatty acid synthetase.

In specific embodiments, the present invention also encompasses aprocess wherein said SCD1 biological activity is measured by an assayselected from among:

-   -   a) SCD1 polypeptide binding affinity;    -   b) SCD1 desaturase activity in microsomes;    -   c) SCD1 desaturase activity in a whole cell assay    -   d) quantification of SCD1 gene expression level; and    -   e) quantification of SCD1 protein level.

Specific embodiments of such an assay may employ a recombinant cell asdisclosed herein.

The present invention also relates to a process wherein the identifiedcompound is further selected from among those compounds that do notsubstantially inhibit in humans the biological activity of delta-5desaturase, delta-6 desaturase or fatty acid synthetase.

In other specific embodiments, the present invention contemplatesemploying SCD1 nucleic acid as disclosed herein and/or SCD1 polypeptideas disclosed herein for use in identifying compounds useful fortreatment of a disorder or condition relating to serum levels oftriglyceride or VLDL.

The assays disclosed herein essentially require the measurement,directly or indirectly, of an SCD1 biological activity. Those skilled inthe art can develop such assays based on well known models, and manypotential assays exist. For measuring whole cell activity of SCD1directly, methods that can be used to quantitatively measure SCDactivity include for example, measuring thin layer chromatographs of SCDreaction products over time. This method and other methods suitable formeasuring SCD activity are well known (Henderson Henderson R J, et al.1992. Lipid Analysis: A Practical Approach. Hamilton S. Eds. New Yorkand Tokyo, Oxford University Press. pp 65-111. ) Gas chromatography isalso useful to distinguish mononunsaturates from saturates, for exampleoleate (18:1) and stearate (18:0) can be distinguished using thismethod. A description of this method is in the examples below. Thesetechniques can be used to determine if a test compound has influencedthe biological activity of SCD1, or the rate at which the SCD introducesa cis-double bond in its substrate palmitate (16:0) or stearate (18:0)to produce palmitolyeoyl-CoA (16:1) or oleyoyl-CoA (18: 1),respectively.

In a preferred embodiment, the invention employs a microsomal assayhaving a measurable SCD1 biological activity. A suitable assay may betaken by modifying and scaling up the rat liver microsomal assayessentially as described by Shimomura et al. (Shimomura, I., Shimano,H., Korn, B. S., Bashmakov, Y., and Horton, J. D. (1998). Tissues arehomogenized in 10 vol. of buffer A (0.1M potassium buffer, pH 7.4). Themicrosomal membrane fractions (100,000×g pellet) are isolated bysequential centrifugation. Reactions are performed at 37° C. for 5 minwith 100 μg of protein homogenate and 60 μM of [1-¹⁴C]-stearoyl-CoA(60,000 dpm), 2 mM of NADH, 0.1M of Tris/HCl buffer (pH 7.2). After thereaction, fatty acids are extracted and then methylated with 10% aceticchloride/methanol. Saturated fatty acid and monounsaturated fatty acidmethyl esters are separated by 10% AgNO₃-impregnated TLC usinghexane/diethyl ether (9:1) as developing solution. The plates aresprayed with 0.2 % 2′,7′-dichlorofluorescein in 95% ethanol and thelipids are identified under UV light. The fractions are scraped off theplate, and the radioactivity is measured using a liquid scintillationcounter.

Specific embodiments of such SCD1 biological activity assay takeadvantage of the fact that the SCD reaction produces, in addition to themonounsaturated fatty acyl-CoA product, H₂O. If 3H is introduced intothe C-9 and C-10 positions of the fatty-acyl-CoA substrate, then some ofthe radioactive protons from this reaction will end up in water. Thus,the measurement of the activity would involve the measurement ofradioactive water. In order to separate the labeled water from thestearate, investigators may employ substrates such as charcoal,hydrophobic beads, or just plain old-fashioned solvents in acid pH.

In a preferred embodiment, screening assays measure SCD1 biologicalactivity indirectly. Standard high-throughput screening assays centre onligand-receptor assays. These may be fluorescence based or luminescencebased or radiolabel detection. Enzyme immunoassays can be run on a widevariety of formats for identifying compounds that interact with SCD1proteins. These assays may employ prompt fluorescence or time-resolvedfluorescence immunoassays which are well known. P³² labeled ATP, istypically used for protein kinase assays. Phosphorylated products may beseparated for counting by a variety of methods. Scintillation proximityassay technology is an enhanced method of radiolabel assay. All thesetypes of assays are particularly appropriate for assays of compoundsthat interact with purified or semi-purified SCD1 protein.

In a preferred embodiment, the assay makes use of ³H-stearoyl CoA (withthe ³H on the 9 and 10 carbon atoms), the substrate for SCD1.Desaturation by SCD1, produces oleoyl CoA and 3H-water molecules. Thereaction is run at room temperature, quenched with acid and thenactivated charcoal is used to separate unreacted substrate from theradioactive water product. The charcoal is sedimented and amount ofradioactivity in the supernatant is determined by liquid scintillationcounting. This assay is specific for SCD1-dependent desaturation asjudged by the difference seen when comparing the activity in wild typeand SCD1-knockout tissues. Further, the method is easily adapted to highthroughput as it is cell-free, conducted at room temperature and isrelatively brief (1 hour reaction time period versus previous period of2 days). This procedure is illustrated more fully in FIGS. 17 to 20.

While the instant disclosure sets forth an effective working embodimentof the invention, those skilled in the art are able to optimize theassay in a variety of ways, all of which are encompassed by theinvention. For example, charcoal is very efficient (>98%) at removingthe unused portion of the stearoyl-CoA but has the disadvantage of beingmessy and under some conditions difficult to pipette. It may not benecessary to use charcoal if the stearoyl-CoA complex sufficientlyaggregates when acidified and spun under moderate g-force. This can betested by measuring the signal/noise ratio with and without charcoalfollowing a desaturation reaction. There are also other reagents thatwould efficiently sediment stearoyl-CoA to separate it from the ³H-waterproduct.

As shown in FIG. 20 (Panel A) the amount of stearoyl-CoA limits thekinetics and magnitude of the ³H-DPM signal monitored as SCD1-dependentdesaturation activity. However, not all of the stearoyl-CoA was consumedby SCD1; >90% remains unavailable to SCD1 either because other enzymespresent in the microsomes (e.g., acyl transferase reactions) utilize itas a substrate and compete with SCD1 and/or stearoyl-CoA is unstableunder the conditions of the experiment. These possibilities may beexamined by monitoring incorporation of the label into phospholipids orby including a buffer mixture (Mg++, ATP and CoA) that would regeneratestearoyl-CoA from stearate and CoA.

As shown in FIG. 20 (Panel B) the assay can be done in a small volumeappropriate for high throughput screening. A preferred embodiment wouldemploy a microcentrifuge satisfactory for spinning 96 well plates.

The following assays are also suitable for measuring SCD1 biologicalactivity in the presence of potential therapeutic agents. These assaysare also valuable as secondary screens to further select SCD1 specificmodulators, inhibitors or agonists from a library of potentialtherapeutic agents.

Cellular based desaturation assays can also be used. By tracking theconversion of stearate to oleate in cells (3T3L1 adipocytes are known tohave high SCD1 expression and readily take up stearate when complexed toBSA) we can evaluate compounds found to be inhibitory in the primaryscreen for additional qualities or characteristics such as whether theyare cell permeable, lethal to cells, and/or competent to inhibit SCD1activity in cells. This cellular based assay may employ a recombinantcell line containing a delta-9 desaturase, preferably hSCD1 (humanSCD1). The recombiant gene is optionally under control of an induciblepromoter and the cell line preferably over-expresses SCD1 protein.

Other assays for tracking other SCD isoforms could be developed. Forexample, rat and mouse SCD2 is expressed in brain. In a preferredembodiment, a microsome preparation is made from the brain as previouslydone for SCD1 from liver. The object may be to find compounds that wouldbe specific to SCD1. This screen would compare the inhibitory effect ofthe compound for SCD1 versus SCD2.

Although unlikely, it is possible that a compound “hit” in the SCD1assay may result from stimulation of an enzyme present in the microsomepreparation that competitively utilizes stearoyl-CoA at the expense ofthat available for SCD1-dependent desaturation. This would mistakenly beinterpreted as SCD1 inhibition. One possibility to examine this problemwould be incorporation into phospholipids of the unsaturated lipid(stearate). By determining effects of the compounds on stimulation ofstearate incorporation into lipids researchers are able to evaluate thispossibility.

Cell based assays may be preferred, for they leave the SCD1 gene in itsnative format. Particularly promising for SCD1 analysis in these typesof assays are fluorescence polarization assays. The extent to whichlight remains polarized depends on the degree to which the tag hasrotated in the time interval between excitation and emission. Since themeasurement is sensitive to the tumbling rate of molecules, it can beused to measure changes in membrane fluidity characteristics that areinduced by SCD1 activity—namely the delta-9 desaturation activity of thecell. An alternate assay for SCD1 involves a FRET assay. FRET assaysmeasure fluorescence resonance energy transfer which occurs between afluorescent molecule donor and an acceptor, or quencher. Such an assaymay be suitable to measure changes in membrane fluidity or temperaturesensitivity characteristics induced by SCD1 biological activity.

The screening assays of the invention may be conducted using highthroughput robotic systems. In the future, preferred assays may includechip devices developed by, among others, Caliper, Inc., ACLARABioSciences, Cellomics, Inc., Aurora Biosciences Inc., and others.

In other embodiments of the present invention, SCD1 biological activitycan also be measured through a cholesterol efflux assay that measuresthe ability of cells to transfer cholesterol to an extracellularacceptor molecule and is dependent on ABCA1 function. A standardcholesterol efflux assay is set out in Marcil et al., Arterioscler.Thromb. Vasc. Biol. 19:159-169, 1999, incorporated by reference hereinfor all purposes.

Preferred assays are readily adapted to the format used for drugscreening, which may consist of a multi-well (e.g., 96-well, 384 well or1536 well or greater) format. Modification of the assay to optimize itfor drug screening would include scaling down and streamlining theprocedure, modifying the labeling method, altering the incubation time,and changing the method of calculating SCD1 biological activity etc. Inall these cases, the SCD1 biological activity assay remains conceptuallythe same, though experimental modifications may be made.

Another preferred cell based assay is a cell viability assay for theisolation of SCD1 inhibitors. Overexpression of SCD decreases cellviability. This phenotype can be exploited to identify inhibitorycompounds. This cytotoxicity may be due to alteration of the fatty acidcomposition of the plasma membrane. In a preferred embodiment, the humanSCD1 cDNA would be placed under the control of an inducible promoter,such as the Tet-On Tet-Off inducible gene expression system (Clontech).This system involves making a double stable cell line. The firsttransfection introduces a regulator plasmid and the second wouldintroduce the inducible SCD expression construct. The chromosomalintegration of both constructs into the host genome would be favored byplacing the transfected cells under selective pressure in the presenceof the appropriate antibiotic. Once the double stable cell line wasestablished, SCD1 expression would be induced using the tetracycline ora tetracycline derivative (eg. Doxycycline). Once SCD1 expression hadbeen induced, the cells would be exposed to a library of chemicalcompounds for HTS of potential inhibitors. After a defined time period,cell viability would then be measured by means of a fluorescent dye orother approach (e.g. turbidity of the tissue culture media). Those cellsexposed to compounds that act to inhibit SCD1 activity would showincreased viability, above background survival. Thus, such an assaywould be a positive selection for inhibitors of SCD1 activity based oninducible SCD1 expression and measurement of cell viability.

An alternative approach is to interfere with the desaturase system. Thedesaturase system has three major proteins: cytochrome b₅, NADH(P)-cytochrome b₅ reductase, and terminal cyanide-sensitive desaturase.Terminal cyanide-sensitive desaturase is the product of the SCD gene.SCD activity depends upon the formation of a stable complex between thethree aforementioned components. Thus, any agent that interferes withthe formation of this complex or any agent that interferes with theproper function of any of the three components of the complex wouldeffectively inhibit SCD activity.

Another type of modulator of SCD1 activity involves a 33 amino aciddestabilization domain located at the amino terminal end of the pre-SCD1protein (Mziaut et al., PNAS 2000, 97: p 8883-8888). It is possible thatthis domain may be cleaved from the SCD1 protein by an as yet unknownprotease. This putative proteolytic activity would therefore act toincrease the stability and half-life of SCD1. Inhibition of the putativeprotease, on the other hand, would cause a decrease in the stability andhalf life of SCD1. Compounds which block or modulate removal of thedestabilization domain therefore will lead to reductions in SCD1 proteinlevels in a cell. Therefore, in certain embodiments of the invention, ascreening assay will employ a measure of protease activity to identifymodulators of SCD1 protease activity. The first step is to identify thespecific protease which is responsible for cleavage of SCD1. Thisprotease can then be integrated into a screening assay. Classicalprotease assays often rely on splicing a protease cleavage site (i.e., apeptide containing the cleavable sequence pertaining to the protease inquestion) to a protein, which is deactivated upon cleavage. Atetracycline efflux protein may be used for this purpose. A chimeracontaining the inserted sequence is expressed in E. coli. When theprotein is cleaved, tetracycline resistance is lost to the bacterium. Invitro assays have been developed in which a peptide containing anappropriate cleavage site is immobilized at one end on a solid phase.The other end is labeled with a radioisotope, fluorophore, or other tag.Enzyme-mediated loss of signal from the solid phase parallels proteaseactivity. These techniques can be used both to identify the proteaseresponsible for generating the mature SCD1 protein, and also foridentifying modulators of this protease for use in decreasing SCD1levels in a cell.

In another aspect, the present invention relates to a process fordetermining the ability of an agent to modulate the activity of a humanstearoyl-CoA desaturase, comprising the steps of:

-   -   (a) contacting the agent under suitable conditions with the        human stearoyl-CoA desaturase of the invention at a        predetermined level of said agent;    -   (b) determining if the activity of said stearoyl-CoA desaturase        changes after said contact,    -   thereby determining if said agent has modulated said activity.

Such an assay may be carried out as a cell free assay employing acellular fractional, such as a microsomal fraction, obtained byconventional methods of differential cellular fractionation, mostcommonly by ultracentrifugation methods. In specific embodiments, suchmodulation may be an increase or decrease in the activity of thedesaturase.

These results suggest that inhibitors of SCD biological activity, suchas hSCD1, in a human, may have the beneficial effect of reducingtriglycerides and/or increasing HDL levels. In addition, increased SCDactivity is also associated with increased body weight index. Thisresult identifies hSCD1 as a useful target for identifying agents formodulating obesity and related conditions. In these human data results,SCD biological activity was measured via the surrogate marker of theratio of 18:1 to 18:0 fatty acids in the total plasma lipid fraction.This marker indirectly measures hSCD1 biological activity.

In a further aspect, the present invention relates to a process fordetermining the ability of an agent to modulate the activity of a humanstearoyl-CoA desaturase in cells expressing the human stearoyl-CoAdesaturase of the invention, comprising the steps of:

-   -   (a) contacting the agent under suitable conditions with a        eukaryotic cell expressing the human stearoyl-CoA desaturase of        the invention at a predetermined level of said agent and under        conditions where said agent may or may not modulate the        expression level of said desaturase;    -   (b) determining if the activity of said stearoyl-CoA desaturase        changes after said contact,    -   thereby determining if said agent has modulated said expression        level.

In specific embodiments of said processes, the modulation may be anincrease or decrease in activity of the desaturase and cells useful inthese processes are preferably mammalian cells, most preferably humancells, and include any of the recombinant cells disclosed herein.

SCD1 Recombinant Cell Lines

In certain embodiments, the present invention contemplates use of a SCD1gene or protein in a recombinant cell line. SCD1 recombinant cell linesmay be generated using techniques known in the art, and those morespecifically set out below.

The present invention also relates to vectors which containpolynucleotides of the present invention, and host cells which aregenetically engineered with vectors of the invention, especially wheresuch cells result in a cell line that can be used for assay of hSCD1activity, and production of SCD1 polypeptides by recombinant techniques.

Host cells are preferably eukaryotic cells, preferably insect cells ofSpodoptera species, most especially SF9 cells. Host cells aregenetically engineered (transduced or transformed or transfected) withthe vectors, especially baculovirus) of this invention which may be, forexample, a cloning vector or an expression vector. Such vectors caninclude plasmids, viruses and the like. The engineered host cells arecultured in conventional nutrient media modified as appropriate foractivating promoters, selecting transformants or amplifying the genes ofthe present invention. The culture conditions, such as temperature, pHand the like, are those previously used with the host cell selected forexpression, and will be apparent to the ordinarily skilled artisan.

The polynucleotides of the present invention may be employed forproducing polypeptides by recombinant techniques. Thus, for example, thepolynucleotide may be included in any one of a variety of expressionvectors for expressing a polypeptide. Such vectors include chromosomal,nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40;bacterial plasmids; phage DNA; baculovirus; yeast plasmids; vectorsderived from combinations of plasmids and phage DNA, viral DNA such asvaccinia, adenovirus, fowl pox virus, and pseudorabies. However, anyother vector may be used as long as it is replicable and viable in thehost.

The appropriate DNA sequence may be inserted into the vector by avariety of procedures. In general, the DNA sequence is inserted into anappropriate restriction endonuclease site(s) by procedures known in theart. Such procedures and others are deemed to be within the scope ofthose skilled in the art.

The DNA sequence in the expression vector is operatively linked to anappropriate expression control sequence(s) (promoter) to direct mRNAsynthesis. As representative examples of such promoters, there may bementioned: LTR or SV40 promoter, the E. coli. lac or trp, the phagelambda P_(L) promoter and other promoters known to control expression ofgenes in prokaryotic or eukaryotic cells or their viruses. Theexpression vector also contains a ribosome binding site for translationinitiation and a transcription terminator. The vector may also includeappropriate sequences for amplifying expression.

In addition, the expression vectors preferably contain one or moreselectable marker genes to provide a phenotypic trait for selection oftransformed host cells such as dihydrofolate reductase or neomycinresistance for eukaryotic cell culture, or such as tetracycline orampicillin resistance in E. coli.

The vector containing the appropriate DNA sequence as hereinabovedescribed, as well as an appropriate promoter or control sequence, maybe employed to transform an appropriate host to permit the host toexpress the protein. Such transformation will be permanent and thus giverise to a cell line that can be used for further testing. Such celllines used for testing will commonly be mammalian cells, especiallyhuman cells.

As representative examples of appropriate hosts, there may be mentionedSpodoptera Sf9 (and other insect expression systems) and animal cellssuch as CHO, COS or Bowes melanoma; adenoviruses; plant cells, and evenbacterial cells, etc, all of which are capable of expressing thepolynucleotides disclosed herein. The selection of an appropriate hostis deemed to be within the knowledge of those skilled in the art basedon the teachings herein. For use in the assay methods disclosed herein,mammalian, especially human, cells are preferred.

More particularly, the present invention also includes recombinantconstructs comprising one or more of the sequences as broadly describedabove. The constructs comprise a vector, such as a plasmid or viralvector, especially where the Baculovirus/SF9 vector/expression system isused, into which a sequence of the invention has been inserted, in aforward or reverse orientation. In a preferred aspect of thisembodiment, the construct further comprises regulatory sequences,including, for example, a promoter, operably linked to the sequence.Large numbers of suitable vectors and promoters are known to those ofskill in the art, and are commercially available. The following vectorsare provided by way of example; Bacterial: pQE70, pQE60, pQE-9 (Qiagen),pBS, pD10, phagescript, psiX174, pBluescript SK, pBSKS, pNH8A, pNH16a,pNH18A, pNH46A (Stratagene); pTRC99a, pKK223-3, pKK233-3, pDR540, pRIT5(Pharmacia); Eukaryotic: pWLNEO, pSV2CAT, pOG44, pXT1, pSG (Stratagene)pSVK3, pBPV, pMSG, pSVL (Pharmacia). However, any other plasmid orvector may be used as long as they are replicable and viable in thehost.

Promoter regions can be selected from any desired gene using CAT(chloramphenicol transferase) vectors or other vectors with selectablemarkers. Two appropriate vectors are pKK232-8 and pCM7. Particular namedbacterial promoters include lacd, lacZ, T3, T7, gpt, lambda P_(R), P_(L)and trp. Eukaryotic promoters include CMV immediate early, HSV thymidinekinase, early and late SV40, LTRs from retrovirus, and mousemetallothionein-I. Selection of the appropriate vector and promoter iswell within the level of ordinary skill in the art.

In a further embodiment, the present invention relates to host cellscontaining the above-described constructs. The host cell can be a highereukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell,such as a yeast cell, or the host cell can be a prokaryotic cell, suchas a bacterial cell. Introduction of the construct into the host cellcan be effected by calcium phosphate transfection, DEAE-Dextran mediatedtransfection, or electroporation (Davis, L., Dibner, M., Battey, I.,Basic Methods in Molecular Biology, (1986)). A preferred embodimentutilizes expression from insect cells, especially SF9 cells fromSpodoptera frugiperda.

The constructs in host cells can be used in a conventional manner toproduce the gene product encoded by the recombinant sequence.Alternatively, the polypeptides of the invention can be syntheticallyproduced by conventional peptide synthesizers.

Mature proteins can be expressed in mammalian cells, yeast, bacteria, orother cells under the control of appropriate promoters. Cell-freetranslation systems can also be employed to produce such proteins usingRNAs derived from the DNA constructs of the present invention.Appropriate cloning and expression vectors for use with prokaryotic andeukaryotic hosts are described by Sambrook, et al., Molecular Cloning: ALaboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989), Wuet al, Methods in Gene Biotechnology (CRC Press, New York, N.Y., 1997),Recombinant Gene Expression Protocols, in Methods in Molecular Biology,Vol. 62, (Tuan, ed., Humana Press, Totowa, N.J., 1997), and CurrentProtocols in Molecular Biology, (Ausabel et al, Eds.,), John Wiley &Sons, NY (1994-1999), the disclosures of which are hereby incorporatedby reference in their entirety.

Transcription of the DNA encoding the polypeptides of the presentinvention by eukaryotic cells, especially mammalian cells, mostespecially human cells, is increased by inserting an enhancer sequenceinto the vector. Enhancers are cis-acting elements of DNA, usually aboutfrom 10 to 300 bp that act on a promoter to increase its transcription.Examples include the SV40 enhancer on the late side of the replicationorigin bp 100 to 270, a cytomegalovirus early promoter enhancer, thepolyoma enhancer on the late side of the replication origin, andadenovirus enhancers.

Generally, recombinant expression vectors will include origins ofreplication and selectable markers permitting transformation of the hostcell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiaeTrp1 gene, and a promoter derived from a highly-expressed gene to directtranscription of a downstream structural sequence. Such promoters can bederived from operons encoding glycolytic enzymes such as3-phosphoglycerate kinase (PGK), α-factor, acid phosphatase, or heatshock proteins, among others. The heterologous structural sequence isassembled in appropriate phase with translation initiation andtermination sequences, and preferably, a leader sequence capable ofdirecting secretion of translated protein into the periplasmic space orextracellular medium. Optionally, the heterologous sequence can encode afusion protein including an N-terminal or C-terminal identificationpeptide imparting desired characteristics, e.g., stabilization orsimplified purification of expressed recombinant product.

Use of a Baculovirus-based expression system is a preferred andconvenient method of forming the recombinants disclosed herein.Baculoviruses represent a large family of DNA viruses that infect mostlyinsects. The prototype is the nuclear polyhedrosis virus (ACMNPV) fromAutographa californica, which infects a number of lepidopteran species.One advantage of the baculovirus system is that recombinantbaculoviruses can be produced in vivo. Following co-transfection withtransfer plasmid, most progeny tend to be wild type and a good deal ofthe subsequent processing involves screening. To help identify plaques,special systems are available that utilize deletion mutants. By way ofnon-limiting example, a recombinant AcMNPV derivative (called BacPAK6)has been reported in the literature that includes target sites for therestriction nuclease Bsu361 upstream of the polyhedrin gene (and withinORF 1629) that encodes a capsid gene (essential for virus viability).Bsf36I does not cut elsewhere in the genome and digestion of the BacPAK6deletes a portion of the ORF1629, thereby rendering the virusnon-viable. Thus, with a protocol involving a system like Bsu361-cutBacPAK6 DNA most of the progeny are non-viable so that the only progenyobtained after co-transfection of transfer plasmid and digested BacPAK6is the recombinant because the transfer plasmid, containing theexogenous DNA, is inserted at the Bsu36I site thereby rendering therecombinants resistant to the enzyme. [see Kitts and Possee, A methodfor producing baculovirus expression vectors at high frequency,BioTechniques, 14, 810-817 (1993). For general procedures, see King andPossee, The Baculovirus Expression System: A Laboratory Guide, Chapmanand Hall, New York (1992) and Recombinant Gene Expression Protocols, inMethods in Molecular Biology, Vol. 62, (Tuan, ed., Humana Press, Totowa,N.J., 1997), at Chapter 19, pp. 235-246.

In accordance with the foregoing, the present invention further relatesto vectors comprising a polynucleotide of the invention and torecombinant eukaryotic cells expressing the stearoyl-CoA desaturase ofthe present invention, preferably wherein said cell is a mammalian cell,most preferably a human cell.

The present invention further relates to processes for using thepolynucleotides, enzymes, and cells disclosed herein in a process fordetermining the ability of an agent to modulate the expression of saidhuman stearoyl-CoA desaturase in cells expressing said humanstearoyl-CoA desaturase of the invention, comprising the steps of:

-   -   (a) contacting the agent under suitable conditions with a        eukaryotic cell expressing the human stearoyl-CoA desaturase of        the invention at a predetermined level of said agent;    -   (b) determining if the expression level of said stearoyl-CoA        desaturase changes after said contact,    -   thereby determining if said agent has modulated said expression        level.

Alternatively, the screening assay may employ a vector constructcomprising the hSCD1 promoter sequence of SEQ ID. No. 3 operably linkedto a reporter gene. Such a vector can be used to study the effect ofpotential transcription regulatory proteins, and the effect of compoundsthat modulate the effect of those regulatory proteins, on thetranscription of SCD1. An example of this type of vector is the pSCD-500plasmid described in the examples below. Reporter gene constructs suchas this are commonly used to indicate whether a test compound hassucceeded in activating the transcription of genes under the control ofa particular promoter.

In specific embodiments, the present invention contemplates a processwherein said modulation is an increase or decrease in said expressionlevel and where said cell may be a mammalian cell, especially a humancell, including any of the recombinant cells disclosed herein. In oneembodiment, the expression level is determined by determining the levelof messenger RNA produced after contact of said cell with said agent.

Factors that may modulate gene expression include transcription factorssuch as, but not limited to, retinoid X receptors (RXRs), peroxisomalproliferation-activated receptor (PPAR) transcription factors, thesteroid response element binding proteins (SREBP-1 and SREBP-2),REV-ERBα, ADD-1, EBPα, CREB binding protein, P300, HNF 4, RAR, LXR, andRORα, NF-Y, C/EBPalpha, PUFA-RE and related proteins and transcriptionregulators. Screening assays designed to assess the capacity of testcompounds to modulate the ability of these transcription factors totranscribe SCD1 are also contemplated by this invention.

Physiological benefits of an increase or decrease in the activity orexpression of hSCD1 include, but are not limited to, decreased plasmatriglycerides and/or increased plasma HDL leading to cardioprotectivebenefit, therapeutic benefit in Type II diabetes, weight loss, improvedgland secretions, and decreased chance of malignancy. Thus, thedetermination of the ability of agents to modulate such activity orexpression affords an opportunity to discover useful therapeutic agentsproducing such effects.

In addition, variations in hSCD1 gene expression, function, stability,catalytic activity and other characteristics may be due to allelicvariations in the polynucleotide sequences encoding such enzymes. Theprocesses disclosed according to the present invention may likewise beused to determine such genomic effects on expression of hSCD1. Using theprocesses of the present invention, such variations may be determined atthe level of DNA polymorphism within the hSCD1 gene and/or promotersequences. Such effects lead to the elucidation of associations betweensuch polymorphisms and predisposition to cancer, neurological disease,skin disease, obesity, diabetes, immune function and lipid metabolismthrough both population and family-based genetic analysis.

Finally, those skilled in the art are able to confirm the relevance ofhSCD1 to human health by analogy to animal models. Well known animaldisease models may be used to ascertain the effect of an hSCD1 modulatoron the growth, development, or disease process in these animals.Additionally, models include genetically modified multicellular animals,such as knock-out or knock-in mice (as detailed in the examples below).

In a general aspect, the present invention relates to a process foridentifying a SCD1-modulating agent, comprising:

-   -   a) contacting under physiological conditions a chemical agent        and a molecule having or inducing SCD1 activity;    -   b) detecting a change in the activity of said molecule having or        inducing SCD1 activity following said contacting;    -   thereby identifying an SCD1 modulating agent.

In specific embodiments of the invention, said molecule having orinducing SCD1 activity is a polypeptide having such activity, or apolynucleotide encoding a polypeptide having such activity, or apolypeptide modulating the activity of a polynucleotide encoding apolypeptide having such activity.

In specific embodiments, said change in activity is an increase inactivity or is a decrease in activity.

In addition, said contacting may be accomplished in vivo. In one suchembodiment, said contacting in step (a) is accomplished by administeringsaid chemical agent to an animal afflicted with a triglyceride (TG)- orvery low density lipoprotein (VLDL)-related disorder and subsequentlydetecting a change in plasma triglyceride level in said animal therebyidentifying a therapeutic agent useful in treating a triglyceride (TG)-or very low density lipoprotein (VLDL)-related disorder. In suchembodiment, the animal may be a human, such as a human patient afflictedwith such a disorder and in need of treatment of said disorder.

In specific embodiments of such in vivo processes, said change in SCD1activity in said animal is a decrease in activity, preferably whereinsaid SCD1 modulating agent does not substantially inhibit the biologicalactivity of a delta-5 desaturase, delta-6 desaturase or fatty acidsynthetase.

In such processes as just disclosed, the detected change in SCD1activity is detected by detecting a change in any, some or all of thefollowing:

-   -   a) SCD1 polypeptide binding affinity;    -   b) SCD1 desaturase activity in microsomes;    -   c) SCD1 desaturase activity in a whole cell;    -   d) SCD1 gene expression; or    -   e) SCD1 protein level.

In accordance with the foregoing, the present invention is also directedto a recombinant cell line comprising a recombinant SCD1 protein asdisclosed herein. In one such embodiment, the whole cell of (c) above isderived from such a cell line, preferably wherein said SCD1 modulatingagent does not substantially inhibit in humans the biological activityof delta-5 desaturase, delta-6 desaturase or fatty acid synthetase.

A recombinant cell line of the invention may also comprise the SCD1promoter nucleic acid sequence of SEQ ID No. 1 operably linked to areporter gene construct. In a specific embodiment thereof, the wholecell of (c) above is derived from a recombinant cell of such a cellline.

In accordance with the disclosure herein, the present invention is alsodirected to an isolated stearoyl-CoA desaturase encoded by thepolynucleotide sequence comprising SEQ ID No. [SCD1 cDNA] as well as areporter gene construct comprising the SCD1 promoter nucleic acidsequence of SEQ ID No. 1 operably linked to a reporter gene,advantageously including usable vectors comprising such the nucleicacids and constructs thereof. Likewise, the present invention alsocontemplates an isolated polypeptide having stearoyl-CoA reductaseactivity and a process as disclosed herein that successfully identifiesa chemical agent that binds to or interacts with such a polypeptide,which process comprises:

-   -   a) contacting such a polypeptide, or a cell expressing such        polypeptide, with a chemical agent; and    -   b) detecting binding or interaction of the chemical agent with        said polypeptide.

In specific embodiments of the process just described, the binding ofthe chemical agent to the polypeptide is detected by a method selectedfrom the group consisting of:

-   -   a) direct detection of chemical agent/polypeptide binding;    -   b) detection of binding by competition binding assay; and    -   c) detection of binding by assay for SCD1 biological activity.

In such processes, modulation of the activity of such polypeptide isdetected by a process comprising contacting the polypeptide or a cellexpressing the polypeptide with a compound that binds to the polypeptidein sufficient amount to modulate the activity of the polypeptide. Inspecific embodiments of this process, the molecule having or inducingSCD1 activity is selected from the group consisting of an SCD1 nucleicacid and/or SCD1 polypeptide as disclosed herein.

In accordance with the foregoing, following identification of chemicalagents having the desired modulating activity, the present inventionalso relates to a process for treating an animal, especially a human,such as a human patient, afflicted with a disease or condition relatingto serum levels of triglyceride or VLDL comprising inhibiting SCD1activity in said human. In a preferred embodiment, said inhibition ofSCD1 activity is not accompanied by substantial inhibition of activityof delta-5 desaturase, delta-6 desaturase or fatty acid synthetase. In aspecific embodiment, the present invention relates to a process fortreating a human patient afflicted with a disorder or condition relatingto serum levels of triglyceride or VLDL comprising administering to saidpatient a therapeutically effective amount of an agent whose therapeuticactivity was first identified by the process of the invention.

In accordance with the foregoing, the present invention also relates toa modulator of SCD1 activity and which is useful in humans for treatmentof a disorder or condition relating to serum levels of triglyceride orVLDL wherein said activity was first identified by its ability tomodulate SCD1 activity, especially where such modulation was firstdetected using a process as disclosed herein according to the presentinvention. In a preferred embodiment thereof, such modulating agent doesnot substantially inhibit fatty acid synthetase, delta-5 desaturase ordelta-6 desaturase of humans.

Thus, the present invention also relates to a process for identifying avertebrate delta-9 stearoyl-CoA desaturase-modulating agent, comprising:

-   -   a) contacting under physiological conditions a chemical agent        and a molecule having or inducing vertebrate delta-9        stearoyl-CoA desaturase activity;    -   b) detecting a change in the activity of said molecule having or        inducing vertebrate delta-9 stearoyl-CoA desaturase activity        following said contacting;    -   thereby identifying a vertebrate delta-9 stearoyl-CoA desaturase        modulating agent.

In a specific embodiment of such process, the contacting in step (a) isaccomplished by administering said chemical agent to an animal afflictedwith a disorder or condition related to serum levels of triglyceride,VLDL, HDL, LDL, total cholesterol, reverse cholesterol transport orproduction or secretion of mucous membranes, monounsaturated fattyacids, wax esters, and like parameters, detecting a change in theactivity of said molecule having or inducing vertebrate delta-9stearoyl-CoA desaturase activity following said contacting and therebyidentifying a therapeutic agent useful in treating a triglyceride, VLDL,HDL, LDL, total cholesterol, reverse cholesterol transport or productionor secretion of mucous membranes, monounsaturated fatty acids, waxesters, and like disease-related disorder.

In accordance with the foregoing, the present invention further relatesto a process for treating a human patient afflicted with a disease orcondition relating to serum levels of triglyceride, VLDL, HDL, LDL,total cholesterol, reverse cholesterol transport or production orsecretion of mucous membranes, monounsaturated fatty acids, wax esters,and like parameters, comprising administering to said human patient atherapeutically effective amount of an agent for which such therapeuticactivity was identified by a process as disclosed herein according tothe invention.

In a preferred embodiments of such process, the modulating agent doesnot substantially inhibit fatty acid synthetase, delta-5 desaturase ordelta-6 desaturase of humans.

Test Compounds/Modulators/Library Sources

In accordance with the foregoing, the present invention also relates totherapeutic and/or diagnostic agents, regardless of molecular size orweight, effective in treating and/or diagnosing and/or preventing any ofthe diseases disclosed herein, preferably where such agents have theability to modulate activity and/or expression of the hSCD1 disclosedherein, and most preferably where said agents have been determined tohave such activity through at least one of the screening assaysdisclosed according to the present invention.

Test compounds are generally compiled into libraries of such compounds,and a key object of the screening assays of the invention is to selectwhich compounds are relevant from libraries having hundreds ofthousands, or millions of compounds having unknown therapeutic efficacy.

Those skilled in the field of drug discovery and development willunderstand that the precise source of test extracts or compounds is notcritical to the screening procedure(s) of the invention. Accordingly,virtually any number of chemical extracts or compounds can be screenedusing the exemplary methods described herein. Examples of such extractsor compounds include, but are not limited to, plant-, fungal-,prokaryotic- or animal-based extracts, fermentation broths, andsynthetic compounds, as well as modification of existing compounds.Numerous methods are also available for generating random or directedsynthesis (e.g., semi-synthesis or total synthesis) of any number ofchemical compounds, including, but not limited to, saccharide-, lipid-,peptide-, and nucleic acid-based compounds. Synthetic compound librariesare commercially available from Brandon Associates (Merrimack, N.H.) andAldrich Chemical (Milwaukee, Wis.). Alternatively, libraries of naturalcompounds in the form of bacterial, fungal, plant, and animal extractsare commercially available from a number of sources, including Biotics(Sussex, UK), Xenova (Slough, UK), Harbor Branch Oceangraphics Institute(Ft. Pierce, Fla.), and PharmaMar, U.S.A. (Cambridge, Mass.). Inaddition, natural and synthetically produced libraries are produced, ifdesired, according to methods known in the art, e.g., by standardextraction and fractionation methods. Furthermore, if desired, anylibrary or compound is readily modified using standard chemical,physical, or biochemical methods.

Thus, in one aspect the present invention relates to agents capable ofmodulating the activity and/or expression of human stearoyl-CoAdesaturase 1 (hSCD1) as disclosed herein, especially where saidmodulating ability was first determined using an assay of comprisinghSCD1or a gene encoding hSCD1, or an assay which measures hSCD1activity. As used herein the term “capable of modulating” refers to thecharacteristic of such an agent whereby said agent has the effect ofchanging the overall biological activity of hSCD1, either by increasingor decreasing said activity, under suitable conditions of temperature,pressure, pH and the like so as to facilitate such modulation to a pointwhere it can be detected either qualitatively or quantitatively andwherein such modulation may occur in either an in vitro or in vivoenvironment. In addition, while the term “modulation” is used herein tomean a change in activity, more specifically either an increase ordecrease in such activity, the term “activity” is not to be limited tospecific enzymatic activity alone (for example, as measured in units permilligram or some other suitable unit of specific activity) but includesother direct and indirect effects of the protein, including increases inenzyme activity due not to changes in specific enzyme activity but dueto changes (i.e., modulation) of expression of polynucleotides encodingand expressing said hSCD1 enzyme. Human SCD1 activity may also beinfluenced by agents which bind specifically to substrates of hSCD1.Thus, the term “modulation” as used herein means a change in hSCD1activity regardless of the molecular genetic level of said modulation,be it an effect on the enzyme per se or an effect on the genes encodingthe enzyme or on the RNA, especially mRNA, involved in expression of thegenes encoding said enzyme. Thus, modulation by such agents can occur atthe level of DNA, RNA or enzyme protein and can be determined either invivo or ex vivo.

In specific embodiments thereof, said assay is any of the assaysdisclosed herein according to the invention. In addition, the agent(s)contemplated by the present disclosure includes agents of any size orchemical character, either large or small molecules, including proteins,such as antibodies, nucleic acids, either RNA or DNA, and small chemicalstructures, such as small organic molecules.

In other aspects, the present invention contemplates agents wherein saidagent is useful in treating, preventing and/or diagnosing a disease orcondition which is identified as being SCD1 related according to thisinvention. Specific embodiments are directed to situations wherein thedisease or condition includes, but is not limited to, serum levels oftriglyceride, VLDL, HDL, LDL, total cholesterol, reverse cholesteroltransport or production of secretions from mucous membranes,monounsaturated fatty acids, wax esters, and the like, cholesteroldisorders, lipidemias, cardiovascular disease, diabetes, obesity,baldness, skin diseases, cancer and multiple sclerosis, especially wherethe disease is a cardiovascular disease or a skin disease or where thecondition is baldness. In a preferred embodiment, such agents willincrease HDL levels in a patient and/or decrease triglyceride levels ina patient. Either or both effects are directly associated with reducedrisk of cardiovascular disease and coronary artery disease.

Some of the known modulators of SCD1 activity include conjugatedlinoleic acid especially trans-10, cis-12 isomer, and thiazoladinedionecompounds such as troglitazone.

While it is envisaged that any suitable mechanism for the inhibition ormodulation of SCD1 activity can be used, three specific examples ofinhibitor classes are envisioned. One class includes those inhibitorsthat effectively inhibit SCD1 expression, such as thiazoladinedionecompounds and polyunsaturated fatty acids. A second class includes thoseinhibitors that effectively inhibit SCD1 enzymatic activity, such asthia-fatty acids, cyclopropenoid fatty acids, and certain conjugatedlinoleic acid isomers. Finally, the third class of inhibitors includesthose agents that are capable of interfering with the proteins essentialto the desaturase system, such as those agents that interfere withcytochrome b₅, NADH (P)-cytochrome b₅ reductase, and terminalcyanide-sensitive desaturase.

For effectively inhibiting the expression of the SCD1 gene, it isenvisioned that any agent capable of disrupting the transcription of theSCD1 gene could be utilized. Since SCD1 is regulated by several knowntranscription factors (e.g. PPAR-γ, SREBP), any agent that affects theactivity of such transcription factors can be used to alter theexpression of the SCD1 gene. One group of such agents includesthiazoladine compounds which are known to activate PPAR-γ and inhibitSCD1 transcription. These compounds include Pioglitazone, Ciglitazone,Englitazone, Troglitazone, and BRL49653. Other inhibitory agents mayinclude polyunsaturated fatty acids, such as linoleic acid, arachidonicacid and dodecahexaenoic acid, which also inhibit SCD1 transcription.

For effectively inhibiting the enzymatic activity of the SCD1 protein,it is envisaged that any agent capable of disrupting the activity of theSCD1 protein could be utilized. For example, certain conjugated linoleicacid isomers are effective inhibitors of SCD1 activity. Specifically,Cis-12, trans-10 conjugated linoleic acid is known to effectivelyinhibit SCD enzyme activity and reduce the abundance of SCD1 mRNA whileCis-9, trans-11 conjugated linoleic acid does not. Cyclopropenoid fattyacids, such as those found in stercula and cotton seeds, are also knownto inhibit SCD activity. For example, sterculic acid(8-(2-octyl-cyclopropenyl)octanoic acid) and Malvalic acid(7-(2-octyl-cyclopropenyl)heptanoic acid) are C18 and C16 derivatives ofsterculoyl- and malvaloyl fatty acids, respectively, having cyclopropenerings at their Δ9 position. These agents inhibit SCD activity byinhibiting Δ9 desaturation. Other agents include thia-fatty acids, suchas 9-thiastearic acid (also called 8-nonylthiooctanoic acid) and otherfatty acids with a sulfoxy moiety.

The known modulators of delta-9 desaturase activity are either not knownto be useful for treating the diseases and disorders linked to SCD1biological activity as claimed in this invention, or else they areotherwise unsatisfactory therapeutic agents. The thia-fatty acids,conjugated linoleic acids and cyclopropene fatty acids (malvalic acidand sterculic acid) are neither useful at reasonable physiologicaldoses, nor are they specific inhibitors of SCD1 biological activity,rather they demonstrate cross inhibition of other desaturases, inparticular the delta-5 and delta-6 desaturases by the cyclopropene fattyacids. These compounds may be useful for establishing control or testmodulators of the screening assays of the invention, but are not subjectto the claims of this invention. Preferred SCD1 modulators of theinvention have no significant or substantial impact on unrelated classesof proteins. In some cases, assays specific for the other proteins, suchas delta-5 and delta-6 activity, will also have to be tested to ensurethat the identified compounds of the invention do not demonstratesignificant or substantial cross inhibition.

The known non-specific inhibitors of SCD1 can be useful in rationaldesign of a therapeutic agent suitable for inhibition of SCD1. All threeinhibitors have various substitutions between carbons #9 and #10;additionally they require conjugation to Co-A to be effective; and areprobably situated in a relatively hydrophobic active site. Thisinformation combined with the known X-ray co-ordinates for the activesite for plant (soluble) SCD can assist the “in silico” process ofrational drug design for therapeutically acceptable inhibitors specificfor SCD1.

This invention also provides an antibody which specifically binds tohuman SCD1 having the amino acid sequence shown in the SwissProtaccession numbers listed above, and which thereby inhibits the activityof SCD1. The instant antibody can be a polyclonal antibody, a monoclonalantibody, or an SCD-binding fragment thereof. In one embodiment, theantibody is isolated, ie.e., an antibody free of any other antibodies.Methods of making and isolating antibodies are well known in the art(Harlow, et al. 1988. Antibodies: A Laboratory Manual; Cold SpringHarbor, N.Y., Cold Spring Harbor Laboratory).

This invention also provides an antisense oligonucleotide whichspecifically binds to human SCD1 mRNA, and which thereby reduces thelevel of SCD1 gene transcription. Methods of making and using antisensemolecules against known target genes are known in the art (Agarwal, S.(1996) Antisense Therapeutics. Totowa, N.J., Humana Press, Inc.)

Combinatorial and Medicinal Chemistry

Typically, a screening assay, such as a high throughput screening assay,will identify several or even many compounds which modulate the activityof the assay protein. The compound identified by the screening assay maybe further modified before it is used in humans as the therapeuticagent. Typically, combinatorial chemistry is performed on the modulator,to identify possible variants that have improved absorption,biodistribution, metabolism and/or excretion, or other importanttherapeutic aspects. The essential invariant is that the improvedcompounds share a particular active group or groups which are necessaryfor the desired modulation of the target protein. Many combinatorialchemistry and medicinal chemistry techniques are well known in the art.Each one adds or deletes one or more constituent moieties of thecompound to generate a modified analog, which analog is again assayed toidentify compounds of the invention. Thus, as used in this invention,therapeutic compounds identified using an SCD1 screening assay of theinvention include actual compounds so identified, and any analogs orcombinatorial modifications made to a compound which is so identifiedwhich are useful for treatment of the disorders claimed herein.

Pharmaceutical Preparations and Dosages

In another aspect the present invention is directed to compositionscomprising the polynucleotides, polypeptides or other chemical agents,including therapeutic, prophylactic or diagnostic agents, such as smallorganic molecules, disclosed herein according to the present inventionwherein said polynucleotides, polypeptides or other agents are suspendedin a pharmacologically acceptable carrier, which carrier includes anypharmacologically acceptable diluent or excipient. Pharmaceuticallyacceptable carriers include, but are not limited to, liquids such aswater, saline, glycerol and ethanol, and the like, including carriersuseful in forming sprays for nasal and other respiratory tract deliveryor for delivery to the ophthalmic system. A thorough discussion ofpharmaceutically acceptable carriers, diluents, and other excipients ispresented in REMINGTON'S PHARMACEUTICAL SCIENCES (Mack Pub. Co., N.J.current edition).

The inhibitors utilized above may be delivered to a subject using any ofthe commonly used delivery systems known in the art, as appropriate forthe inhibitor chosen. The preferred delivery systems include intravenousinjection or oral delivery, depending on the ability of the selectedinhibitor to be adsorbed in the digestive tract. Any other deliverysystem appropriate for delivery of small molecules, such as skinpatches, may also be used as appropriate.

In another aspect the present invention further relates to a process forpreventing or treating a disease or condition in a patient afflictedtherewith comprising administering to said patient a therapeutically orprophylactically effective amount of a composition as disclosed herein.

Diagnosis & Pharmacogenomics

In an additional aspect, the present invention also relates to a processfor diagnosing a disease or condition in a patient, commonly a humanbeing, suspected of being afflicted therewith, or at risk of becomingafflicted therewith, comprising obtaining a tissue sample from saidpatient and determining the level of activity of hSCD1 in the cells ofsaid tissue sample and comparing said activity to that of an equalamount of the corresponding tissue from a patient not suspected of beingafflicted with, or at risk of becoming afflicted with, said disease orcondition. In specific embodiments thereof, said disease or conditionincludes, but is not limited to, cholesterol disorders, lipidemias,cardiovascular disease, diabetes, obesity, baldness, skin diseases,cancer and multiple sclerosis, especially wherein said disease is acardiovascular disease or a skin disease or said condition is baldness.

In an additional aspect, this invention teaches that hSCD1 haspharmacogenomic significance. Variants of hSCD1 including SNPs (singlenucleotide polymorphisms), cSNPs (SNPs in a cDNA coding region),polymorphisms and the like may have dramatic consequences on a subject'sresponse to administration of a prophylactic or therapeutic agent.Certain variants may be more or less responsive to certain agents. Inanother aspect, any or all therapeutic agents may have greater or lesserdeleterious side-effects depending on the hSCD1 variant present in thesubject.

In general, the invention discloses a process of selecting aprophylactic and/or therapeutic agent for administration to a subject inneed thereof comprising,

-   -   (a) determining at least a part of the hSCD1 nucleic acid        sequence of said subject; and    -   (b) comparing said hSCD1 nucleic sequence to known variants of        hSCD1 nucleic acids;    -   wherein said known variants are correlated with responsiveness        to said agent and said agent is selected for said subject on the        basis of a desired correlation. In this method the correlation        may be a prophylactic and/or therapeutic effect or it may be        avoidance of a deleterious side effect, or any other desired        correlation.

In a pharmacogenomic application of this invention, an assay is providedfor identifying cSNPs (coding region small nucleotide polymorphisms) inhSCD1 of an individual which are correlated with human disease processesor response to medication. The inventors have identified two putativecSNPs of hSCD1 to date:In exon 1, a C/A cSNP at nt 259, corresponding toa D/E amino acid change at position 8; and

in exon 5, a C/A cSNP at nt 905, corresponding to a UM amino acid changeat position 224. (Sequence numbering according to GenBank Accession:AF097514). It is anticipated that these putative cSNPs may be correlatedwith human disease processes or response to medication of individualswho contain those cSNPs versus a control population. Those skilled inthe art are able to determine which disease processes and whichresponses to medication are so correlated.

In carrying out the procedures of the present invention it is of courseto be understood that reference to particular buffers, media, reagents,cells, culture conditions and the like are not intended to be limiting,but are to be read so as to include all related materials that one ofordinary skill in the art would recognize as being of interest or valuein the particular context in which that discussion is presented. Forexample, it is often possible to substitute one buffer system or culturemedium for another and still achieve similar, if not identical, results.Those of skill in the art will have sufficient knowledge of such systemsand methodologies so as to be able, without undue experimentation, tomake such substitutions as will optimally serve their purposes in usingthe methods and procedures disclosed herein.

In applying the disclosure, it should be kept clearly in mind that otherand different embodiments of the methods disclosed according to thepresent invention will no doubt suggest themselves to those of skill inthe relevant art.

EXAMPLE 1 Disruption of Stearoyl-CoA Desaturase1 Gene in Mice CausesDecreased Plasma Triglycerides Levels, as Well as other Defects in LipidMetabolism

This example identifies, for the first time, specific SCD1 biologicalactivities in mouse by characterizing an SCD1 gene specific knock-outmouse.

To investigate the physiological functions of SCD, we have generatedSCD1 null (SCD1−/−) mice. The lipoprotein profile of SCD1 null(knock-out) mice demonstrates a striking decrease in triglyceride (i.e.,VLDL) levels while maintaining approximately normal HDL and LDL levels.This result confirms that a mutation in SCD1 is a causative mutation ofa low triglyceride (TG) lipoprotein profile in mice, and is distinctfrom other SCD isoforms in the mouse in this regard. Due to the severityof this phenotype it is clear that other SCD isoforms are unlikely toaffect TG levels to such a great extent.

Targeted Disruption of the SCD1 Gene

FIG. 1A shows the strategy used to knock out the SCD1 gene. The mouseSCD1 gene includes 6 exons. The first 6 exons of the gene were replacedby a neomycin-resistant cassette by homologous recombination, resultingin the replacement of the complete coding region of the SCD1 gene (FIG.1A). The vector was electroporated into embryonic stem cells and theclones that integrated the neo cassette were selected by growth ongeneticin. Targeted ES clones were injected into C57BI/6 blastocystsyielding four lines of chimeric mice that transmitted the disruptedallele through the germ-line. The mutant mice were viable and fertileand bred with predicted Mendelian distributions. A PCR based screen toassay successful gene targeting of the SCD1 locus is shown in FIG. 1B.To determine whether the expression of the SCD1 gene was ablated weperformed Northern blot analysis (FIG. 1C) SCD1 mRNA is undetectable inliver of SCD1−/− mice and reduced by approximately 50% in SCD+/− mice.SCD2 mRNA was expressed at low levels in both SCD1−/− mice and wild-typemice. Consistent with Northern blot results, Western blot analysisshowed no immunoreactive SCD protein in liver from SCD−/− mice, whereasSCD1 protein was detectable in both heterozygous and wild-type livertissue in a manner dependent on gene dosage. SCD enzyme activity inliver, as measured by the rate of conversion of [1-¹⁴C]stearoyl-CoA to[1-¹⁴C]oleate (FIG. 1E) was high in the wild-type mice but wasundetectable in the total extracts of livers of the SCD1−/− mice.

Lipid Analysis

Analysis of liver cholesterol ester (0.8±0.1 vs. 0.3±0.1 mg/g liver) andliver triglycerides (12.6±0.3 vs. 7.5±0.6 mg/g liver) showed that SCD1KO animals have lower amounts of both cholesterol esters andtriglycerides than wild-type controls. Plasma lipoprotein analysisshowed a decrease in plasma triglycerides (120.6±6.8 vs. 45.4±3.8) inSCD−/− mice compared to normal controls. These findings are similar tofindings in asebia mice. FIG. 2 records the plasma lipoprotein profileobtained using fast performance liquid chromatography. SCD1 Knock-Outmice showed a 65% reduction of triglyceride in VLDL fraction; but littleor no significant difference in LDL or HDL levels.

Asebia mice are compared with the SCD1 Knock-Out mice in FIG. 2. Thefindings are remarkably similar. Asebia mice plasma lipoproteins wereseparated by fast performance liquid chromatography and the distributionof triglycerides among lipoproteins in the various density fractions ofthe mice (n=3) are shown. FIG. 3 shows an additional example of anAsebia mouse lipoprotein profile. These profiles showed a majordifference in the distribution of triglycerides in the VLDL fraction ofthe SCD−/− and SCD−/+ mice. The levels of triglycerides in the SCD−/+were 25 mg/dl in the VLDL, with very low levels in the LDL and HbLfractions. In contrast the SCD−/− had very low levels of triglyceridesin the three lipoprotein fractions.

Fatty Acid Analysis

We also determined the levels of monounsaturated fatty acids in varioustissues. Table 1 shows the fatty acid composition of several tissues inwild-type and SCD−/− mice. The relative amounts of palmitoleate(16:1n-7) in liver and plasma from SCD−/− mice decreased by 55% and 47%while those of oleate (18:1n-9) decreased by 35% and 32%, respectively.The relative amount of palmitoleate in white adipose tissue and skin ofSCD−/− mice were decreased by more than 70%, whereas the reduction ofoleate in these tissues was less than 20% although the reduction wassignificant statistically. These changes in levels of monounsaturatedfatty acids resulted in reduction of desaturation indices indicatingreduction in desaturase activity. In contrast to these tissues, thebrain, which expresses predominantly the SCD2 isoform, had a similarfatty acid composition and unaltered desaturation index in both wildtype and SCD−/− mice. We conclude that SCD1 plays a major role in theproduction of monounsaturated fatty acids in the liver.

TABLE I Fatty acid composition of several tissues from SCD1 knockoutmice 14:0 16:0 16:1n-7 18:0 18:1n-9 18:1n-7 18:2n-6 20:0 20:1n-9 20:1n-7Liver +/+ 0.8 25.9 1.1 16.1 16.2 1.6 16.3 0.0 0.0 0.0 −/− 1.0 27.2 0.522.8 10.6 1.0 13.9 0.0 0.0 0.0 Eyelid +/+ 1.3 15.0 2.4 9.3 19.6 3.4 5.25.2 24.1 9.6 −/− 1.9 22.4 1.5 20.3 16.1 3.7 6.8 7.5 3.7 7.4 WAT +/+ 3.327.6 5.2 5.7 35.1 1.9 19.5 0.0 0.0 0.0 −/− 2.7 29.2 1.5 14.8 29.1 1.718.7 0.0 0.0 0.0 Skin +/+ 3.5 29.3 4.0 9.7 32.4 2.1 15.0 0.0 0.0 0.0 −/−3.1 30.7 1.4 14.2 28.1 1.8 17.6 0.0 0.0 0.0 Brain +/+ 1.1 25.7 0.8 21.616.5 3.1 1.1 0.0 0.0 0.0 −/− 1.1 26.2 0.8 21.1 15.8 3.3 1.2 0.0 0.0 0.0Eye Ball +/+ 2.9 31.3 1.6 28.5 19.1 2.6 0.0 0.0 0.0 0.0 −/− 3.2 32.3 1.529.8 18.8 2.4 0.0 0.0 0.0 0.0 20:4n-6 22:6n-3 16:1/16:0 18:1n-9/18:018:1n-7/18:0 20:1n-9/20:0 20:1n-7/20:0 Liver +/+ 9.2 7.8 0.041 1.0060.100 0.000 0.000 −/− 6.8 8.8 0.018 0.465 0.044 0.000 0.000 Eyelid +/+0.9 0.8 0.160 2.108 0.366 4.635 1.846 −/− 1.7 1.5 0.067 0.793 0.1820.493 0.987 WAT +/+ 0.3 0.2 0.190 6.211 0.340 0.000 0.000 −/− 0.4 0.80.050 1.967 0.115 0.000 0.000 Skin +/+ 0.9 0.7 0.136 3.351 0.219 0.0000.000 −/− 0.9 0.8 0.045 1.982 0.128 0.000 0.000 Brain +/+ 9.4 12.4 0.0320.764 0.145 0.000 0.000 −/− 9.5 13.1 0.030 0.752 0.154 0.000 0.000 EyeBall +/+ 2.9 7.2 0.051 0.671 0.091 0.000 0.000 −/− 2.3 5.8 0.046 0.6320.081 0.000 0.000 Tissue total lipids from each mouse were extracted.The lipids were methyl esterified and quantified by GLC as describedunder Experimental Procedures. Standard errors for all values were lessthan 25% of the mean and were omitted from table for clarity. Boldvalues represent a statistical significance of p < 0.05 betweenwild-type and SCD −/− mice (student's t test). The values ofmonounsaturated/saturated fatty acids were calculated from the meanvalue.

FIG. 4 (quantified in Table 2) demonstrate that SCD1 is a majorcontributor to the plasma desaturation indices (ratio of plasma18:1/18:0 or 16:1/16:0 in the total lipid fraction), as judged by plasmafatty acid analysis of both the SCD1 KO and asebia mice. In both animalmodels, a reduction of approximately 50% or greater is observed in theplasma desaturation indices. This demonstrates that the plasmadesaturation index is highly dependent on the function of SCD1

TABLE 2 Fatty acid desaturation indices in asebia mutants andheterozygotes Sex/genotype 18:1/18:0 16:1/16:0 Male +/− 1.393 0.044 Male−/− 0.732 0.018 Female +/− 1.434 0.074 Female −/− 0.642 0.021 Female +/−1.203 0.081 Female −/− 0.574 0.022

Experimental Procedures for Knockout Mice:

Generation of the SCDI Knockout Mice.

Mouse genomic DNA for the targeting vector was cloned from 129/SVgenomic library. The targeting vector construct was generated byinsertion of a 1.8-kb Xba I/Sac I fragment with 3′ homology as a shortarm and 4.4-kb Cla I/Hind III fragment with 5′ homology cloned adjacentto neo expression cassette. The construct also contains a HSV thymidinekinase cassette 3′ to the 1.8-kb homology arm, allowingpositive/negative selection. The targeting vector was linearized by NotI and electroporated into embryonic stem cells. Selection with geneticinand gancyclovior was performed. The clones resistant to both geneticinand gancyclovior were analyzed by Southern blot after EcoRI restrictionenzyme digestion and hybridized with a 0.4-kb probe located downstreamof the vector sequences. For PCR genotyping, genomic DNA was amplifiedwith primer A

5′-GGGTGAGCATGGTGCTCAGTCCCT-3′ (SEQ ID NO: 2)

which is located in exon 6, primer B

5′-ATAGCAGGCATGCTGGGGAT-3′ (SEQ ID NO: 3)

which is located in the neo gene (425 bp product, targeted allele), andprimer C

5′-CACACCATATCTGTCCCCGACAAATGTC-3′ (SEQ ID NO: 4)

which is located in downstream of the targeting gene (600 bp product,wild-type allele). PCR conditions were 35 cycles, each of 45 sec at 94°C., 30 sec at 62° C., and 1 min at 72° C. The targeted cells weremicroinjected into C57BI/6 blastocysts, and chimeric mice were crossedwith C57BLU6 or 129/SvEv Taconic females, and they gave the germ-linetransmission. Mice were maintained on a 12-h dark/light cycle and werefed a normal chow diet, a semi-purified diet or a diet containing 50% (%of total fatty acids) triolein, tripalmitolein or trieicosenoin. Thesemi-purified diet was purchased from Harlan Teklad (Madison, Wis.) andcontained: 20% vitamin free casein, 5% soybean oil, 0.3% L-cystine,13.2% Maltodextrin, 51.7% sucrose, 5% cellulose, 3.5% mineral mix(AIN-93G-MX), 1.0% vitamin mix (AIN-93-VX), 0.3% choline bitartrate. Thefatty acid composition of the experimental diets was determined by gasliquid chromatography. The control diet contained 11% palmitic acid(16:0), 23% oleic acid (18:1n-9), 53% linoleic acid (18:2n-6) and 8%linolenic acid (18:3n-3). The high triolein diet contained 7% 16:0, 50%18:1n-9, 35% 18:2n-6 and 5% 18:3n-3.

Materials

Radioactive [-³²P]dCTP (3000 Ci/mmol) was obtained from Dupont Corp.(Wilmington, Del.). Thin layer chromatography plates (TLC Silica GelG60) were from Merck (Darmstadt, Germany). [1-¹⁴C]-stearoyl-CoA waspurchased from American Radiolabeled Chemicals, Inc. (St. Louis, Mo.).Immobilon-P transfer membranes were from Millipore (Danvers, Mass.). ECLWestern blot detection kit was from Amersham-Pharmacia Biotech, Inc.(Piscataway, N.J.). All other chemicals were purchased from Sigma (St.Louis, Mo.).

Lipid Analysis

Total lipids were extracted from liver and plasma according to themethod of Bligh and Dyer (Bligh and Dyer, 1959), and phospholipids, waxesters, free cholesterol, triglycerides and cholesterol esters wereseparated by silica gel high performance TLC. Petroleum hexane/diethylether/acetic acid (80:30:1) or benzene/hexane (65:35) was used as adeveloping solvent (Nicolaides and Santos, 1985). Spots were visualizedby 0.2% 2′,7′-dichlorofluorecein in 95% ethanol or by 10% cupric sulfatein 8% phosphoric acid. The wax triester, cholesterol ester andtriglyceride spots were scraped, 1 ml of 5% HCl-methanol added andheated at 100° C. for 1 h (Miyazaki et al., 2000). The methyl esterswere analyzed by gas-liquid chromatography using cholesterolheptadecanoate, triheptadecanoate and heptadecanoic acid as internalstandard. Free cholesterol, cholesterol ester and triglycerides contentsof eyelid and plasma were determined by enzymatic assays (Sigma St.Louis, Mo. and Wako Chemicals, Japan).

Isolation and Analysis of RNA

Total RNA was isolated from livers using the acidguanidinium-phenol-chloroform extraction method (Bemlohr et al., 1985).Twenty micrograms of total RNA was separated by 1.0% agarose/2.2 Mformaldehyde gel electrophoresis and transferred onto nylon membrane.The membrane was hybridized with 32P-labeled SCD1 and SCD2 probes. pAL15probe was used as control for equal loading (Miyazaki, M., Kim, Y. C.,Gray-Keller, M. P., Attie, A. D., and Ntambi, J. M. (2000). Thebiosynthesis of hepatic cholesterol esters and triglycerides is impairedin mice with a disruption of the gene for stearoyl-CoA desaturase 1. JBiol Chem 275, 30132-8).

SCD Activity Assay

Stearoyl-CoA desaturase activity was measured in liver microsomesessentially as described by Shimomura et al. (Shimomura, I., Shimano,H., Kom, B. S., Bashmakov, Y., and Horton, J. D. (1998). Nuclear sterolregulatory element-binding proteins activate genes responsible for theentire program of unsaturated fatty acid biosynthesis in transgenicmouse liver. J Biol Chem 273, 35299-306. ). Tissues were homogenized in10 vol. of buffer A (0.1M potassium buffer, pH 7.4). The microsomalmembrane fractions (100,000×g pellet) were isolated by sequentialcentrifugation. Reactions were performed at 37° C. for 5 min with 100 μgof protein homogenate and 60 μM of [1-¹⁴C]-stearoyl-CoA (60,000 dpm), 2mM of NADH, 0.1M of Tris/HCl buffer (pH 7.2). After the reaction, fattyacids were extracted and then methylated with 10% aceticchloride/methanol. Saturated fatty acid and monounsaturated fatty acidmethyl esters were separated by 10 % AgNO₃-impregnated TLC usinghexane/diethyl ether (9:1) as developing solution. The plates weresprayed with 0.2 % 2′,7′-dichlorofluorescein in 95% ethanol and thelipids were identified under UV light. The fractions were scraped offthe plate, and the radioactivity was measured using a liquidscintillation counter. The enzyme activity was expressed as nmole min⁻¹mg⁻¹ protein.

Immunoblotting

Pooled liver membranes from 3 mice of each group were prepared asdescribed by Heinemann et al (Heinemann and Ozols, 1998). The sameamount of protein (25 μg) from each fraction was subjected to 10%SDS-polyacrylamide gel electrophoresis and transferred to Immobilon-Ptransfer membranes at 4° C. After blocking with 10% non-fat milk in TBSbuffer (pH 8.0) plus Tween at 4° C. overnight, the membrane was washedand incubated with rabbit anti-rat SCD as primary antibody and goatanti-rabbit IgG-HRP conjugate as the secondary antibody. Visualizationof the SCD protein was performed with ECL western blot detection kit.

Histology

Tissues were fixed with neutral-buffered formalin, embedded in paraffin,sectioned and stained with hematoxylin and eosin.

This work was supported by a grant-in-aid from the American HeartAssociation-Wisconsin affiliate and in part by grant #DAMD17-99-9451from DOD.

Experimental Procedures for Asebia Mice:

Animals and Diets-Asebia homozygous (ab J/ab J or −/−) and heterozygous(+/ab J or +/−) mice were obtained from the Jackson Laboratory (BarHarbor, Me.) and bred at the University of Wisconsin Animal CareFacility. In this study, comparisons are made between the homozygous(−/−) and the heterozygous (+/−) mice since the latter areindistinguishable from normal mice. Mice were housed in a pathogen-freebarrier facility operating a 12-h light/12-h dark cycle. At 3 weeks ofage, these mice were fed ad libitum for 2 wks or 2 months, on laboratorychow diet or on a semi-purified diet containing 50% (% of total fattyacids) triolein or tripalmitolein. The semi-purified diet was purchasedfrom Harlan Teklad (Madison, Wis.) and contained: 18% vitamin freecasein, 5% soybean oil, 33.55% corn starch, 33.55% sucrose, 5%cellulose, 0.3%-L methionine, 0.1% choline chloride, salt mix (AIN-76A)and vitamin mix (AIN-76A). The fatty acid composition of theexperimental diets was determined by gas liquid chromatography. Thecontrol diet contained 11% palmitic acid (16:0), 23% oleic acid(18:1n-9), 53% linoleic acid (18:2n-6) and 8% linoleic acid (18:3n-3).The high triolein diet contained 7% 16:0, 50% 18:1n-9, 35% 18:2n-6 and5% 18:3n-3. The high trpalmitolein diet contained 6% 16:0, 49%palmitoleic acid (16:1n-7), 12% 18:1n-9, 27% 18:2n-6 and 4% 18:3n-3.

Animals were anesthetized at about 10:00 a.m. by intraperitonealinjection of pentobarbital sodium (0.08 mg/g of body weight) Nembutal,Abbot, North Chicago, Ill.). Liver was isolated immediately, weighed,and kept in liquid nitrogen. Blood samples were obtained from theabdominal vein.

Materials-Radioactive α-³ ²P]dCTP (3000 Ci/mmol) was obtained fromDupont Corp. (Wilmington, Del.). Thin layer chromatography plates (TLCSilica Gel G60) were from Merck (Darmstadt, Germany).[1-¹⁴C]-stearoyl-CoA, [³H]cholesterol and [1-¹⁴C]oleoyl-CoA werepurchased from American Radiolabeled Chemicals, Inc. (St. Louis, Mo.).Immobilon-P transfer membranes were from Millipore (Danvers, Mass.). ECLWestern blot detection kit was from Amersham-Pharmacia Biotech, Inc.(Piscataway, N.J.). LT-1 transfection reagent was from PanVera (Madison,Wis.). All other chemicals were purchased from Sigma (St. Louis, Mo.).The antibody for rat liver microsome SCD was provided by Dr. Juris Ozolsat University of Connecticut Health Center. pcDNA3-1 expression vectorSCD1 was provided by Dr. Trabis Knight at Iowa state university.

Lipid Analysis-Total lipids were extracted from liver and plasmaaccording to the method of Bligh and Dyer (Bligh, E. G., and Dyer, W. J.(1959) Can J Biochem Physiol 37, 911-917. ), and phospholipids, freecholesterol, triglycerides and cholesterol esters were separated bysilica gel TLC. Petroleum ether/diethyl ether/acetic acid (80:30:1) wasused as a developing solvent. Spots were visualized by 0.2%2′,7′-dichlorofluorecein in 95% ethanol or by 10% cupric sulfate in 8%phosphoric acid. The phospholipid, cholesterol ester and triglyceridespots were scraped, 1 ml of 5% HCl-methanol added and heated at 100° C.for 1 h. The methyl esters were analyzed by gas-liquid chromatographyusing cholesterol heptadecanoate as internal standard (Lee, K. N.,Pariza, M. W., and Ntambi, J. M. (1998) Biochem. Biophys. Res. Commun.248, 817-821; Miyazaki, M., Huang, M. Z., Takemura, N., Watanabe, S.,and Okuyama, H. (1998) Lipids 33, 655-61). Free cholesterol, cholesterolester and triglycerides contents of liver and plasma were determined byenzymatic assays (Sigma St. Louis, Mo. and Wako Chemicals, Japan).

Plasma Lipoprotein Analysis-Mice were fasted a minimum of 4 hours andsacrificed by CO 2 asphyxiation and/or cervical dislocation. Blood wascollected aseptically by direct cardiac puncture and centrifuged(13,000×g, 5 min, 4° C.) to collect plasma. Lipoproteins werefractionated on a Superose 6HR 10/30 FPLC column (Pharmacia). Plasmasamples were diluted 1:1 with PBS, filtered (Cameo 3AS syringe filter,0.22 μm) and injected onto the column that had been equilibrated withPBS containing 1 mM EDTA and 0.02% NaN 3. The equivalent of 100 μl ofplasma was injected onto the column. The flow rate was set constant at0.3 ml/min. 500 μl fractions were collected and used for totaltriglyceride measurements (Sigma). Values reported are for totaltriglyceride mass per fraction. The identities of the lipoproteins havebeen confirmed by utilizing anti-ApoB immunoreactivity for LDL andAnti-Apo A1 immnunoreactivity for HDL (not shown).

EXAMPLE 2 Demonstration of Significant Correlation Between the 18:1/18:0FFA Ratio and TG/HDL Levels in Humans

This example demonstrates, for the first time, that delta-9 desaturaseactivity in humans correlates directly with serum levels of triglyceride(VLDL) and inversely with serum HDL level and total serum cholesterol.

Experimental Design:

Plasma from a total of 97 individuals was analyzed for fatty acidcontent by gas chromatography (GC). Total free fatty acid (FFA) contentwas measured and the ratios of oleate to stearate (18:1/18:0) andpalmitoleate to palmitate (16:1/16:0) were computed, defined as thedesaturation indices, as above. We sought to find a relationship betweenthese ratios and three clinical indicators; plasma TG (triglyceride)levels, plasma HDL (high density lipoprotein) levels, and total plasmacholesterol.

Patient Sample:

The patient sample was chosen to maximize phenotypic diversity in termsof HDL. Within our cohort, 21 individuals displayed a high HDL phenotype(>90^(th) percentile for age and sex), 12 individuals displayed a lowHDL phenotype of unknown etiology (<5^(th) percentile for age and sex),while six displayed a low HDL phenotype due to mutations in the ABCA1gene. 33 individuals fall within normal HDL parameters (<90^(th) and>5^(th) percentile for age and sex).

We also attempted to diversity our sample in terms of TG levels, byincluding 9 individuals with Familial Combined Hyperlipidemia (FCHL) whohave high TG and/or cholesterol as well as 16 additional controlindividuals with normal TG levels.

In some cases, multiple individuals from the same family were tested.Five of the six individuals with an ABCA1 mutation are part of the samefamily (NL-020). Multiple individuals were also tested from otherpedigrees segregating a low HDL phenotype that is not genetically linkedto ABCA1. In this category, two affected individuals were tested fromNL-008, while four affected individuals were tested from NL-001. Theremaining six individuals with a low HDL phenotype are not related toone another, and were chosen from distinct pedigrees. Of thoseindividuals with high HDL, seven of them were unrelated to one another.It is not yet clear if the high HDL observed in these individuals has aclear genetic basis in family members. The remaining 14 individuals witha high HDL phenotype, six of them are from family HA-1 and eight arefrom a distinct family, HA-3. Unaffected individuals related to thosewith both low and high HDL were also tested.

Our cohort show wide variation in TG and HDL levels. In general,individuals with low HDL have high TG levels and those with high TGlevels tend to have low HDL levels. This relationship between TG and HDLhas been previously noted in the literature (Davis et al., 1980).

Analysis of fatty acid esters was determined as follows. Cells frompatient samples were washed twice with cold phosphate-buffered salineand total cellular lipids were extracted three times with CHCl3/MeOH(2:1 v/v). The three lipid extractions were combined in a screw-cappedglass tube, dried under N2 gas at 40° C. in a heat block, andresuspended in toluene. Fatty acid methyl esters were produced fromBCl3/MeOH (Alltech, Deerfield Ill.), extracted with hexane, dried, andresuspended in hexane. Fatty acid methyl esters were identified using aHewlett-Packard 6890 gas chromatograph equipped with a 7683 autoinjector and an HP-5 column (30 m′ 0.25 mm, 0.25 μm film thickness)connected to a flame ionization detector set at 275° C. The injector wasmaintained at 250° C. The column temperature was held at 180° C. for 2min following injection, increased to 200° C. at 8° C./min, held at 200°C. for 15 min, and then increased to 250° C. at 8° C./min. Under theseconditions, the ?9?16:1?, 16:0?, ?9?18:1- and 18:0-methyl esters elutedat 9.2 min, 9.7 min, 15.3 min, and 16.4 min, respectively. See Lee etal. (1998). Biochem. Biophys. Res. Commun. 248:817-821; Miyazaki et al.(1998) Lipids 33:655-661; and Miyazaki M, Kim Y C, Gray-Keller M P,Attie A D, Ntambi J M. 2000. J Biol Chem. 275(39):30132-8.

Results

Linear regression analysis was carried out using the entire human dataset. The ratio of 18:1/18:0 showed a significant relationship to TGlevels (r²=0.39, p<0.0001) (FIG. 5 a), as well as significantcorrelations to HDL levels (r²=0.12, p=0.0006) (FIG. 5 b).

The 16:1/16:0 plasma fatty acid ratio was measured in a similar manner,although the results were not as striking. A weak relationship betweenthe relative level of 16:1/16:0 to plasma TG levels was observed(r²=0.05, p=0.03) (FIG. 6), whereas the relationship between the16:1/16:0 ratio and HDL levels did not reach significance (not shown).In contrast to the 18:1/18:0 ratio, the 16:1/16:0 ratio did explain aportion of the variance in total cholesterol levels (r²=0.06,p=0.02)(not shown).

Overall the 18:1/18:0 ratio accounted for 18% of the variance in totalplasma fatty acid content (p=0.005) while the 16:1/16:0 ratio accountedfor 8% of the variation in this value (p=0.02), when the individualswith FCHL and their associated controls were excluded from the analysis(not shown in the Figure).

Finally, for the portion of our sample for which Body Mass Index (BMI)values were available, we measured a positive correlation between18:1/18:0 ratios and BMI (r²=0.13, p=0.00) (data not shown).

The sample was stratified based on HDL levels to determine if therelationship between SCD activity (as measured by the 18:1/18:0 ratio)and TG levels was independent of the primary cause of the observeddyslipidemia.

A positive correlation was observed between 18:1/18:0 and TG in personswith high HDL.

Analysis of those individuals with a high HDL phenotype (>90^(th)percentile) demonstrated a significant relationship between the18:1/18:0 ratio and TG levels (r²=0.40, p<0.005) (FIG. 7). Therelationship between 18:1/18:0 ratio and HDL levels in this group didnot reach significance (data not shown). The 16:1/16:0 ratio did notaccount for a significant proportion of the variance in totalcholesterol, TG or HDL levels in this subset of our cohort.

In order to determine if a stronger relationship between the 18:1/18:0index and TG levels would be apparent in a genetically homogenousbackground, the HA-1 and HA-3 families were analyzed separately. Bothaffected and unaffected family members were included in the analysis. Inboth families, a similar relationship between 18:1/18:0 and TG levelswas observed (HA-1: r²=0.36, p=0.005 (FIG. 8 a), HA-3: r²=0.32, p=0.009(not shown in the figure)). The strength of these relationships wassimilar to that observed in the entire cohort. 18:1/18:0 ratios alsocorrelated with HDL levels in HA-1, although this relationship did notreach significance in HA-3 (HA-1: r²=0.32, p=0.009 (FIG. 8 b), HA-3:r²=0.10, p=0.22 (not shown)).

A positive correlation was also observed between 18:1/18:0 and TG inthose with low HDL. When all individuals with low HDL (<5^(th)percentile) were analyzed as a group, a significant relationship wasobserved between the 18:1/18:0 ratio and TG levels (r²=0.49, p=0.0009)(FIG. 9). As observed in our analysis of the high HDL patient subset,the relationship between the 18:1/18:0 ratio and HDL did not meetsignificance in the low HDL group (data not shown). In addition, nosignificant result was noted when the 16:1/16:0 ratio was regressed withHDL, TG and total cholesterol values.

Analysis of family NL-001, which segregated a low HDL phenotype ofunknown genetic etiology, and family NL-0020, which segregated an ABCA1mutation, tended towards the relationships noted above between fattyacid ratios and lipid parameters when affected individuals in eachfamily were considered. However, these results did not reach statisticalsignificance due to the small number of individuals analyzed in eachcase (NL-001: FIG. 10 a, b and NL0020: FIG. 11 a, b). A general trendtowards higher 18:1/18:0 ratios in older Tangiers patients was noted.This could be an effect independent of the disease, although an agedependent effect on the 18:1/18:0 ratio was noted in neither of the HA-1and HA-3 families nor in the entire cohort (not shown in FIG. 11).

FIG. 12 shows the relationship between the 18:1/18:0 ratio and TG levels(r2=0.56, p=0.03) (FIG. 12 a), HDL levels (r2=0.64, p=0.009) (FIG. 12 b)and total cholesterol levels (r2=0.50, p=0.03) in persons with FamilialCombined Hyperlipidemia (FCHL) (FIG. 12 c).

Our analysis is the first demonstration in humans that SCD function, asmeasured by the 16:1/16:0 and 18:1/18:0 desaturation indices, correlatespositively with plasma TG levels and inversely with plasma HDL.Importantly, we observe this correlation irrespective of the underlyingcause of hyper- or hypo-triglyceridemia, suggesting that therelationship between SCD activity and TG levels is a generalized effect.Therefore, inhibition of SCD activity in humans is linked to decreasedserum TG (or VLDL) levels, increased total cholesterol levels, increasedHDL levels, and decreased body-mass-index (BMI), independent of theprimary cause of TG elevation. Importantly, SCD1 inhibitors could beused as a combination therapy in patients also being treated for FCHL.

In summary, when taken together, Examples 1 and 2 establish for thefirst time a positive correlation between SCD1 activity and TG levels inmammals, as well as an inverse correlation between SCD1 activity and HDLin humans. Our analysis of the asebia and SCD1 KO are definitive intheir implication of SCD1 as the major contributor to the desaturationindex. We have used this index as a surrogate for SCD1 activity in ourhuman studies. Thus, inhibitors of SCD1 function in mammals, includinghumans, are likely to both lower TG and raise HDL.

EXAMPLE 3 Plasma Fatty Acid Analysis in a Mouse Model of Dyslipidemia

In order to confirm the above described relationship observed in humansbetween the 18:1/18:0 desaturation index and TG levels. We alsoperformed plasma fatty acid analysis in a mouse model of the humandisease FCHL. In the mouse hyperlipidemic strain (“hyplip”) TG levelsare elevated as compared to wild-type.

The hyperlipidemic mouse HcB-19 showed an elevated 18:1/18:0desaturation index. This mouse model of familial combined hyperlipidemia(HcB-19) displays elevated levels of TG, cholesterol, as well asincreased secretion of VLDL and apoB (Castellani et al, Mapping a genefor combined hyperlipidaemia in a mutant mouse strain. Nat Genet;18(4):374-7 (1998).

Plasma fatty acid analysis demonstrated that these animals have asignificantly elevated 18:1/18:0 ratio when compared to unaffectedcontrols of the parental strain (FIG. 13). The HcB-19 animals did not,however, show a significant elevation of the 16:1/16:0 index whencompared to controls. Therefore, we observe a positive correlationbetween the 18:1/18:0 desaturation index and TG levels in this animalmodel of FCHL.

EXAMPLE 4 Transcriptional Regulators of SCD1 and Their Use as DrugScreening Targets

This example reports, for the first time, the complete genomic promotersequence of human SCD1. This promoter is used herein to identifyregulatory elements that modulate and control SCD1 expression in humans,and identifies regulatory proteins that are suitable targets for smallmolecule intervention to modulate expression of SCD1 in humans.

The human SCD1 promoter sequence is set forth at SEQ ID No. 1. Thissequence has not been accurately annotated in Genbank and has beenreported as 5′UTR in a number of records.

FIG. 14 illustrates the location of regulatory sequences and bindingsites in the homologous region of the mouse SCD1 and human SCD1 promoterand 5′-flanking regions. The top scale denotes the position relative tothe transcriptional start site. Important promoter sequence elements areindicated.

The human SCD1 promoter structure is similar to that of the mouse SCD1isoform and contains conserved regulatory sequences for the binding ofseveral transcription factors, including the sterol regulatory elementbinding protein (SREBP), CCMT enhancer binding protein-alpha (C/EBPa)and nuclear factor-1 (NF-1) that have been shown to transactivate thetranscription of the mouse SCD gene. Cholesterol and polyunsaturatedfatty acids (PUFAs) decreased the SCD promoter-luciferase activity whentransiently transfected into HepG2 cells. The decrease in promoteractivity in the reporter construct correlated with decreases inendogenous SCD mRNA and protein levels. Transient co-transfection intoHepG2 cells of the human SCD promoter-luciferase gene construct togetherwith expression vector for SREBP revealed that SREBP trans-activates thehuman SCD promoter. Our studies indicate that like the mouse SCD1 gene,the human SCD gene is regulated by polyunsaturated fatty acids andcholesterol at the level of gene transcription and that SREBP plays arole in the transcriptional activation of this gene.

Construction of the Chimeric Promoter Luciferase Plasmid

A human placenta genomic library in bacteria-phage I EMBL3 was screenedwith a 2.0 kb PstI insert of the mouse pC3 cDNA (Ntambi, J. M., Buhrow,S. A., Kaestner, K. H., Christy, R. J., Sibley, E., Kelly, T. J. Jr.,and M. D. Lane. 1988. Differentiation-induced gene expression in 3T3-L1preadipocytes: Characterization of a differentially expressed geneencoding stearoyl-CoA desaturase. J. Biol. Chem. 263: 17291-17300. ) asa radioactive probe and seven plaques were isolated. Two of theseplaques were purified to homogeneity, the DNA isolated and designatedHSCD1and HSCD3. A DNA primer based on the sequence corresponding to thefirst exon of the cDNA of the published human stearoyl-CoA desaturasegene (Zhang, L., G. E. Lan, S. Parimoo, K. Stenn and S. M. Proutey.1999. Human stearoyl-CoA desaturase alternative transcripts generatedfrom a single gene by usage of tandem polyadenylation sites. Biochem. J.340: 255-264) was synthesized and used to sequence the two phage clonesby the dideoxy nucleotide chain termination method. A preliminarysequence was generated and primers upstream

5′NNNNGGTACCTTNNGAAAAGAACAGCGCCC 3′ SEQ ID No. 5

and downstream:

5′NNNNAGATCTGTGCGTGGAGGTCCCCG 3′ SEQ ID No. 6

were designed to amplify approximately 540 bases of the promoter regionupstream of the transcription start site: These primers contain insertedrestriction enzyme sites (underlined), Kpn1 for upstream, and BgIII fordownstream, with a 4 base overhang region to allow restriction enzymedigestion. PCR was then performed on the phage clones and the amplified500 bp fragment was isolated from a 1% agarose gel.

The amplified fragment was digested with Kpn1 and BgIII and then clonedinto the Kpn1 and BgIII sites of the pGL3 basic vector (Promega) thatcontains the luciferase reporter gene and transformed into DH5 competentE. coli cells. Plasmid DNA was purified on Qiagen columns and sequencedby the dideoxynucleotide chain termination method using as primerscorresponding to DNA sequences within the multiple cloning site butflanking the inserted DNA. The SCD promoter luciferase gene constructthat was generated was designated as pSCD-500.

Isolation and Analysis of RNA—Total RNA was isolated from HepG2 cellsusing the acid guanidinium-phenol-chloroform extraction method. Twentymicrograms of total RNA was separated by 0.8% agarose/2.2 M formaldehydegel electrophoresis and transferred onto nylon membrane. The membranewas hybridized with ³²P-labeled human SCD cDNA probe generated by PCR asfollows: pAL15 probe was used as control for equal loading.

Immunoblotting—Cell extracts were prepared from HepG2 cells that hadbeen treated with the various fatty acids or cholesterol as described byHeinemann et al (17). The same amount of protein (60 μg) from eachfraction was subjected to 10% SDS-polyacrylamide gel electrophoresis andtransferred to Immobilon-P transfer membranes at 4° C. After blockingwith 10% non-fat milk in TBS buffer (pH 8.0) plus 0.5% Tween at 4° C.overnight, the membrane was washed and incubated with rabbit anti-ratSCD as primary antibody (17) and goat anti-rabbit IgG-HRP conjugate asthe secondary antibody. Visualization of the SCD protein was performedwith ECL western blot detection kit.

Effect of Cholesterol, Polyunsaturated Fatty Acids and Arachidonic Acidon the Expression of hSCD1

Cell Culture and DNA transfections—HepG2 cells, were grown in LowGlucose DMEM supplemented with 10% Fetal Bovine Serum and 1%Penicillin/Streptomycin solution and maintained at 37 C, 5% CO₂ in ahumidified incubator. Cells were passaged into 6 cm dishes to give40-70% confluence in about 12-16 hours. Cells were then transfected with5 μg plasmid DNA per plate of pSCD-500 or the Basic PGL3 reporter aswell as well as the pRL-TK, internal controls (Promega) using the LT-1transfection reagent (Pan Vera). After 48 hours, cells were rinsed withPBS and then treated in Williams' E Media, a fatty acid-free media,containing insulin, dexamethasone, and appropriate concentrations ofalbumin-conjugated fatty acids as indicated in figures and legends.Cells were also treated with ethanol alone (as control) or cholesterol(10 μg/mL) and 25-OH cholesterol (1 μg/mL) dissolved in ethanol. Afteran additional 24 h, extracts were prepared and assayed for luciferaseactivity. Non-transfected cells were used as the blank and RenillaLuciferase was used as an internal control. Cell extracts were assayedfor protein according to Lowry, and all results were normalized toprotein concentration as well as to renilla luciferase counts. Eachexperiment was repeated at least three times, and all data are expressedas means ±SEM.

Results:

The sequence of the amplified promoter region of the SCD1 gene is shownat SEQ ID. No. 1.

When compared to the mouse SCD1 promoter sequence, it was found thatseveral functional regulatory sequences identified in the mouse SCD1promoter are absolutely conserved at the nucleotide level and also withrespect to their spacing within the proximal promoters of the two genes(FIG. 14). Both the TTAATA homology, the C/EBPa and NF-1 are in the samelocations in both the mouse SCD1 and human promoters. Further upstreamthe sterol regulatory element (SRE) and the two CCMT box motifs that arefound in the polyunsaturated fatty acid responsive element (PUFA-RE) ofthe mouse SCD1 and SCD2 promoters. The spacing of these elements isconserved in the three promoters.

We tested whether the human SCD gene expression was also repressed bycholesterol and polyunsaturated fatty acids. Human HepG2 cells werecultured and then treated with 100 μM arachidonic acid, DHA or 10 μg/mlcholesterol and 1 μg/ml of 25-hydroxycholesterol cholesterol as we havedescribed previously. Total mRNA was isolated and subjected to northernblot analysis using a probe corresponding to the human cDNA andgenerated by the PCR method using primers based on published human SCDcDNA sequence. FIG. 15 shows that M, DHA and cholesterol decreased thehuman SCD mRNA expression in a dose dependent manner. The western blotof the protein extracts of the cells treated with PUFAs and cholesterolshows that PUFAs and cholesterol decreased the levels of the SCD proteinas well (data not shown).

To assess the possible effect of SREBP binding on the activity of thehuman SCD promoter the human luciferase promoter construct wasco-transfected in HepG2 cells together with an expression vectorcontaining SREBP1a. After 72 h, extracts of the transfected cells wereassayed for luciferase activity. Data were normalized to cell extractexpressing the Renilla luciferase as an internal control. As shown infigure SREBP transactivates the promoter in a dose dependent mannergiving rise to an increase up to 40-fold. This experiment shows thatSREBP plays a role in regulating the human SCD gene.

Published reports indicated that the mature form of SREBP, in additionto activating the lipogenic genes, also mediates PUFA and cholesterolrepression of lipogenic genes, including mouse SCD1. To observe theregulatory effects of mature SREBP-1a and PUFAs on the activity of SCDpromoters, HepG2 hepatic cells were transiently co-transfected with 20ng (per 6-cm dish) of plasmid DNA containing the human SCD promoter asdescribed above but this time the transfections were carried out in thepresence of cholesterol to inhibit the maturation of the endogenousSREBP and thus ensure that there was little mature form of theendogenous SREBP present in the cells. After transfection, the cellswere then treated with, arachidonic acid, EPA and DHA as albumincomplexes and luciferase activity was then assayed using a luminometer.If SREBP mediates PUFA repression of the human SCD gene, SCD promoteractivity would not diminish upon treatment the transfected cells withPUFA. However addition of M, EPA or DHA continued to repress SCDpromoter activity with only a slight attenuation (data not shown). Thus,SREBP maturation does not seem to exhibit the selectivity required toexplain PUFA control of SCD gene transcription suggesting that PUFA mayutilize a different protein in addition to the SREBP to repress humanSCD gene transcription.

These results establish that hSCD1 is transcriptionally regulated bySREBP, NF-Y, C/EBPalpha, PUFA-RE and alternate proteins andtranscription regulators. Each one of these proteins is therefore be anattractive drug screening target for identifying compounds whichmodulate SCD1 expression in a cell; and thereby being useful fortreating the human diseases, disorders and conditions which are taughtby the instant invention.

EXAMPLE 5

The SCD1 Knock-Out Displays Cutaneous and Ocular Abnormalities.

To investigate the physiological functions of SCD, we have generatedSCD1 knock-out (SCD1−/−) mice. We found that the levels of C16:1 weredramatically decreased in the tissues of SCD1−/− mice whereas a dramaticdecrease in C18:1 was noted only in liver where SCD1 alone and not SCD2is normally expressed. In tissues such as the eyelid, adipose and skinwhere both SCD1 and SCD2 are expressed, 18:1was only slightly decreased.The monounsaturated fatty acids levels of the brain and eyeball which donot express SCD1 were unchanged. The liver and skin of the SCD−/− micewere deficient in cholesterol esters and triglycerides while the, eyelidin addition was deficient in eyelid-specific wax esters of long chainmonounsaturated fatty acids mainly C20:1. In addition the eyelid of theSCD−/− mice had higher levels of free cholesterol. The SCD−/− miceexhibited cutaneous abnormalities with atrophic sebaceous gland andnarrow eye fissure with atrophic meibomian glands which is similar tothe dry eye syndrome in humans. These results indicate that SCD1deficiency can affect the synthesis not only of monounsaturated fattyacids as components of tissue cholesterol ester and triglycerides butother lipids such as wax esters of the eyelid.

Gross Pathology and Histolgical Examination of SCD−/− Knock-Out Mice.

SCD−/− mice were healthy and fertile but they have cutaneousabnormalities. These abnormalities started around weaning age (3-4weeks) with dry skin, fine epidermal scaling, and hair loss which becamemore severe with aging. In addition, the mice exhibited narrow eyefissures. Pathological examination of the skin and eyelids showed thewild-type mice had a prominent and well-differentiated sebaceous andmeibomian glands (data not shown). On the other hand, skin and eyelid ofSCD−/− appeared atrophic acinar cells in the sebaceous and meibomianglands (data not shown). No abnormalities were found in cornea andretina (data not shown).

SCD−/− Mice have Low Levels of Eyelid and Skin Neutral Lipids

We measured free cholesterol (FC) and cholesterol ester (CE),triglycerides and wax ester contents in the eyelid. Thin layerchromatography (TLC) of lipids extracted from eyelid of SCD1−/− micedemonstrated markedly reduced cholesterol ester and triglyceride and waxester levels compared to the lipids extracted from eyelid of wild-typemice (FIG. 16A). Table 3 compares eyelid lipid contents between SCD−/−and wild type mice.

TABLE 3 Genotype +/+ −/− Cholesterol ester content (mg/g eyelid) 18.1 ±0.7 4.8 ± 0.3 Free cholesterol content (mg/g eyelid)  5.3 ± 0.5 8.4 ±0.2 Wax triester ((mg/g eyelid) 36.8 ± 4.4 10.3 ± 0.8  Triglycerides(mg/g eyelid) 13.8 ± 0.6 5.5 ± 0.4Each value of the table denotes the mean ±SD (n=4). All mice were 6weeks old and fed a chow diet. Bold values of the −/− column denote astatistical significance (p<0.01) between the wild type and SCD−/− mice.

As shown in Table 3, and shows that the cholesterol ester content ineyelid and skin of SCD1−/− mice was decreased by 74%, while freecholesterol increased by 1.75-fold. There was a reduction in the CE andtriglyceride level in the liver of the SCD−/− as well but there was nodifference in free cholesterol content in liver (data not shown). Thetriglyceride and wax ester contents in the eyelid of the SCD−/− micedecreased by 60% and 75%, respectively.

FIG. 16B shows use of a different solvent according to Nicolaids et al(Nicolaides, N., and Santos, E. C. (1985). The di- and triesters of thelipids of steer and human meibomian glands. Lipids 20, 454-67)consisting of hexane/benzene (45:65) was used to resolve the differentwax esters shows that the triester is the major wax ester. Thesetriesters as well as the diesters were decreased by 72% in the SCD−/−mice. The eyelid of wax triester content decreased by 72% in the SCD−/−mice. Similar to eyelid, cholesterol ester and triglyceride contents inthe skin of SCD−/− mice decreased by 43% and 53%, respectively whilefree cholesterol increased by 1.9-fold (Table 3, and FIGS. 16A and B).Finally, the absolute monounsaturated fatty acid content in eachfraction was dramatically reduced in the SCD1−/− mice with correspondingincreases in the saturated fatty acids (data not shown).

Dietary 18:1 Did Not Restore Abnormalities of Skin and Eyelid in SCD−/−Mice

Oleate is one of the most abundant fatty acids in the diet. The cellularmonounsaturated fatty acids used for cholesterol ester and triglyceridesynthesis, could be synthesized either de novo by Fatty Acid Synthaseand SCD or by incorporation of exogenous oleate indirectly from thediet. To determine whether dietary oleate could substitute for theendogenously synthesized oleate and restore the hair, skin and eyeabnormalities of the SCD−/− mice, we supplemented the semi-purifiedmouse diets with high levels of 18:1n-9 (50% of total fat) as triolein,and then fed these diets to SCD−/− mice for 2 weeks. However, theseabnormalities were not restored by these diets which contained highmonounsaturated fatty acids. This suggests that SCD1 specific inhibitorswould act to reduce TG levels regardless of diet. Instead, cholesterolester, wax ester and triglyceride levels in the eyelids of SCD−/− micefed with high 18:1n-9 were still lower than those of SCD+/+ mice (datanot shown), suggesting that endogenously synthesized monounsaturatedfatty acids are required for the synthesis of the cholesterol esterstriglycerides and wax esters of mebum.

In the present study, we have established SCD1 null mice and have shownthat SCD deficiency caused substrate-selective and tissue-selectiveexpression. The level of palmitoleate in SCD−/− mice is decreased bygreater than 50% in all tissues including liver, which expressed SCD1 inwild-type mice. On the other hand, the alternations of oleate level weretissue-specific.

Similar to asebia mice which have a spontaneous mutation of SCD1, SCD−/−mice exhibited abnormalities of hair growth, skin, and eye with completepenetrance. These phenotypes were noticeable from weaning age.Histological examination of the skin and eye lid showed that the atropicsebaceous gland in the skin and meibomian gland in the border of eyelidwhere SCD1 is abundantly expressed, lacked sebaceous and meibomiansecreted lipids, the so-called, sebum and mebum, respectively. In fact,we found that neutral lipids including triglyceride, several kinds ofwax esters and cholesterol ester which are known to be components ofsebum and mebum, were markedly reduced in the eyelid and also from theepidermis (data not shown) of the SCD−/− mice.

Chronic blepharitis similar to the eye lid abnormalities we havedescribed in the SCD−/− mice, is one of the most common frustratingdisease in humans. Shine and McCulley (Shine, W. E., and McCulley, J. P.(1998). Keratoconjunctivitis sicca associated with meibomian secretionpolar lipid abnormality. Arch Ophthalmol 116, 849-52) have reported thatchronic blephatitis may be due to lipid abnormalities in mebum. Thenature of these lipid abnormalities were not characterized in detail.They however, found that mebum from patients with meibomiankeratoconjunctivitis have decreased levels of oleic acid, a majorproduct of SCD whereas that from patients with meibomian seborrhea haveincreased levels of 18:1. These observations, together with our presentstudy, suggest that the alternation of SCD activity can be implicated inchronic blepharitis. Thus, the SCD may become a potential target for thedevelopment of therapeutic and preventive drugs for the treatment of eyediseases.

Promoter Sequence of Human Stearoyl-CoA Desaturase 1

SEQ ID No. 1 ggtccccgcc ccttccagag agaaagctcc cgacgcggga tgccgggcagaggcccagcg gcgggtggaa gagaagctga gaaggagaaa cagaggggag ggggagcgaggagctggcgg cagagggaac agcagattgc gccgagccaa tggcaacggc aggacgaggtggcaccaaat tcccttcggc caatgacgag ccggagttta cagaagcctc attagcatttccccagaggc aggggcaggg gcagaggccg ggtggtgtgg tgtcggtgtc ggcagcatccccggcgccct gctgcggtcg ccgcgagcct cggcctctgt ctcctccccc tcccgcccttacctccacgc gggaccgccc gcgccagtca actcctcgca ctttgcccct gcttggcagcggataaaagg gggctgagga aataccggac acggtcaccc gttgccagctctagccttta aattcccggc tcggggacct ccacgcaccg cggctagcgc cgacaaccagctagcgtgca aggcgccgcg gctcagcgcg taccggcggg cttcgaaacc gcagtcctccggcgaccccg aactccgctc cggagcctca gccccct

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1. A method for identifying an agent that modulates plasma triglyceridelevels in a mammal comprising administering to a mammal an agent thathas Stearoyl-coenzyme A-desaturase (SCD1)-modulating activity in an invitro microsome-based assay, wherein said agent tested in said assaydoes not comprise coenzyme A and does not substantially inhibit Δ-5 orΔ-6 -desaturase and detecting a change in plasma triglyceride level insaid mammal as a result of said administering thereby identifying saidagent as a plasma triglyceride modulating agent.
 2. A method foridentifying an agent that modulates plasma triglyceride levels in amammal comprising administering to a mammal an agent that hasStearoyl-coenzyme A-desaturase (SCD1)-modulating activity in an in vitrocell-based assay wherein said agent tested in said assay does notcomprise coenzyme A and does not substantially inhibit Δ-5 or Δ-6desaturase and detecting a change in plasma triglyceride level in saidmammal as a result of said administering thereby identifying said agentas a plasma triglyceride modulating agent.
 3. The method of claim 1,wherein said agent that has SCD1-modulating activity was firstidentified as having SCD1-modulating activity using said in vitromicrosome-based assay.
 4. The method of claim 1, wherein said plasmatriglyceride modulating activity is a decrease in plasma triglyceridelevel.
 5. The method of claim 1, wherein said mammal is a human.
 6. Themethod of claim 2, wherein said agent that has SCD1-modulating activitywas first identified as having SCD1- modulating activity using said invitro cell-based assay.
 7. The method of claim 2, wherein said plasmatriglyceride modulating activity is a decrease in plasma triglyceridelevel.
 8. The method of claim 2, wherein said mammal is a human.